TW201800411A - Substituted tricyclic herteocyclic compound and use thereof - Google Patents
Substituted tricyclic herteocyclic compound and use thereof Download PDFInfo
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- TZBXJXINYLUDDF-UHFFFAOYSA-N CC[n]1nc(CN(C(CC2)CCN2C(C)C)c2c3c-4n[nH]c3ccn2)c-4c1 Chemical compound CC[n]1nc(CN(C(CC2)CCN2C(C)C)c2c3c-4n[nH]c3ccn2)c-4c1 TZBXJXINYLUDDF-UHFFFAOYSA-N 0.000 description 3
- GBFJCNVQMLXWJW-UHFFFAOYSA-N CC(C)N(CC1)CCC1N(Cc1n[n](C)cc1-1)c2c3c-1n[nH]c3ccn2 Chemical compound CC(C)N(CC1)CCC1N(Cc1n[n](C)cc1-1)c2c3c-1n[nH]c3ccn2 GBFJCNVQMLXWJW-UHFFFAOYSA-N 0.000 description 2
- FTBQLTCAFBRMKD-UHFFFAOYSA-N CC(C)N(CC1)CCC1N(CCCc1n[nH]2)c3c1c2ccn3 Chemical compound CC(C)N(CC1)CCC1N(CCCc1n[nH]2)c3c1c2ccn3 FTBQLTCAFBRMKD-UHFFFAOYSA-N 0.000 description 1
- HRIIPKGSZKKGJL-UHFFFAOYSA-N CC(C)[n]1nc(CN(C(CC2)CCN2C(C)C)c2c3c-4n[nH]c3ccn2)c-4c1 Chemical compound CC(C)[n]1nc(CN(C(CC2)CCN2C(C)C)c2c3c-4n[nH]c3ccn2)c-4c1 HRIIPKGSZKKGJL-UHFFFAOYSA-N 0.000 description 1
- VSNSFTQDGATYDS-UHFFFAOYSA-N CC(C)[n]1nc(CN(C2CCN(C)CC2)c2c3c-4n[nH]c3ccn2)c-4c1 Chemical compound CC(C)[n]1nc(CN(C2CCN(C)CC2)c2c3c-4n[nH]c3ccn2)c-4c1 VSNSFTQDGATYDS-UHFFFAOYSA-N 0.000 description 1
- AOAAIUCYRQKVJY-UHFFFAOYSA-N CC(C)[n]1nc(CN(C2CCOCC2)c2c3c-4n[nH]c3ccn2)c-4c1 Chemical compound CC(C)[n]1nc(CN(C2CCOCC2)c2c3c-4n[nH]c3ccn2)c-4c1 AOAAIUCYRQKVJY-UHFFFAOYSA-N 0.000 description 1
- XGHQONHLGXFBDP-UHFFFAOYSA-N CCN(CCN1C2CCOCC2)c2n[nH]c3ccnc1c23 Chemical compound CCN(CCN1C2CCOCC2)c2n[nH]c3ccnc1c23 XGHQONHLGXFBDP-UHFFFAOYSA-N 0.000 description 1
- BGJUMLIDKBULTP-UHFFFAOYSA-N CC[n]1nc(CN(C2CCOCC2)c2c3c-4n[nH]c3ccn2)c-4c1 Chemical compound CC[n]1nc(CN(C2CCOCC2)c2c3c-4n[nH]c3ccn2)c-4c1 BGJUMLIDKBULTP-UHFFFAOYSA-N 0.000 description 1
- JUOVVSMMEKTBSF-UHFFFAOYSA-N CN(CC1)CCC1N(Cc1c2cccc1)c(nccc1N)c1C2=N Chemical compound CN(CC1)CCC1N(Cc1c2cccc1)c(nccc1N)c1C2=N JUOVVSMMEKTBSF-UHFFFAOYSA-N 0.000 description 1
- RYXRIEBJEADDKW-UHFFFAOYSA-N C[n]1nc(CN(C2CCOCC2)C(NCC=C2N)=C2C2=N)c2c1 Chemical compound C[n]1nc(CN(C2CCOCC2)C(NCC=C2N)=C2C2=N)c2c1 RYXRIEBJEADDKW-UHFFFAOYSA-N 0.000 description 1
- YATFNRRBIGOWSE-UHFFFAOYSA-N FC(c1cc(-c2n[nH]c3ccnc(N(C4)C5CCOCC5)c23)c4cn1)(F)F Chemical compound FC(c1cc(-c2n[nH]c3ccnc(N(C4)C5CCOCC5)c23)c4cn1)(F)F YATFNRRBIGOWSE-UHFFFAOYSA-N 0.000 description 1
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Abstract
Description
相關申請的交叉引用 本申請要求於2016年6月15日向國家智慧財產權局提交的申請號為PCT/CN2016/085811的國際專利申請的優先權和權益，通過引用將其全部內容併入本文。 CROSS- REFERENCE TO RELATED APPLICATIONS RELATED APPLICATIONS RELATED APPLICATIONS RELATED APPLICATIONS RELATED APPLICATIONS
本發明屬於製藥技術領域，更具體地涉及化合物、包括其組合物和二者用於治療由癌症和神經退行性疾病引起的病症的用途。特別地，本文提供了能夠抑制一種以上的激酶，特別是LRRK，更特別是LRRK2的取代的三環雜環化合物。The present invention is in the field of pharmaceutical technology, and more particularly relates to the use of compounds, including compositions thereof, and both for the treatment of conditions caused by cancer and neurodegenerative diseases. In particular, provided herein are substituted tricyclic heterocyclic compounds capable of inhibiting more than one kinase, particularly LRRK, more particularly LRRK2.
抗帕金森氏病（PD）是第二常見的神經退行性疾病，影響1-2%的老年人群[1]。全基因組關聯分析（GWAS）在非家族性PD的24個位點具有28種相關的遺傳風險變異[2]。其中，發現LRRK2（Park8）中的突變也是以遺傳形式，確定了在家族性和非家族性PD中驅動發病機制的共用分子途徑，並且包括最常見的疾病原因[3,4]。PD致病性LRRK2突變圖主要與激酶（G2019S，I2020T）和ROC-COR結構域（R1441C/G/H，Y1699C）有關，表示這些酶促活性對發病機制至關重要[5]。致病性突變的頻率在整體2%左右是罕見的[6,7]，然而在某些種族人群的高達40%的患者中發現了這種最常見突變G2019S，其將該激酶啟動2至3倍[8-13]。除了致病性突變外，LRRK2中常見的遺傳變異性是散發性PD的危險因素[14-16]。Anti-Parkinson's disease (PD) is the second most common neurodegenerative disease affecting 1-2% of the elderly population [1]. Genome-wide association analysis (GWAS) has 28 associated genetic risk variants at 24 sites of non-familial PD [2]. Among them, mutations in LRRK2 (Park8) were also found to be genetically defined to share a common molecular pathway that drives pathogenesis in both familial and non-familial PD, and include the most common causes of disease [3, 4]. The PD pathogenic LRRK2 mutation map is mainly related to the kinase (G2019S, I2020T) and ROC-COR domains (R1441C/G/H, Y1699C), indicating that these enzymatic activities are critical for pathogenesis [5]. The frequency of pathogenic mutations is rare at around 2% overall [6,7], however, the most common mutation, G2019S, was found in up to 40% of patients in certain ethnic groups, which initiated the kinase 2 to 3 Times [8-13]. In addition to pathogenic mutations, the genetic variability common in LRRK2 is a risk factor for sporadic PD [14-16].
2004年，LRRK2被確定為負責與PARK8基因座相關的對PD遺傳的基因[17,18]，並發現其由51個外顯子組成，產生較大的（268 kDa）蛋白質。隨後，確定了LRRK2一級結構中的許多變體，包括還出現在散發性PD中的與家族性PD分離的顯性突變，以及增加散發性PD發展的終生風險的LRRK2基因座處的多態性[20-22]。In 2004, LRRK2 was identified as a gene responsible for PD inheritance associated with the PARK8 locus [17,18] and was found to consist of 51 exons, resulting in a larger (268 kDa) protein. Subsequently, many variants of the primary structure of LRRK2 were identified, including dominant mutations that were also found in sporadic PD and familial PD, and polymorphisms at the LRRK2 locus that increased lifetime risk of sporadic PD development. [20-22].
LRRK2是其核心包含兩種酶促功能的多結構域蛋白。由以稱為Roc結構域的C-末端（COR）終止的間隔結構域的複合蛋白（ROC）的Ras組成的GTP酶結構域緊接著屬於絲氨酸/蘇氨酸激酶的激酶結構域。這種酶促核心被在LRRK2 N末端的包含犰狳蛋白、錨蛋白和富含亮氨酸重複（LRR）結構域的蛋白質-蛋白質相互作用結構域所包圍[23]。LRRK2 C末端含有WD40結構域，這被認為是蛋白質折疊所必需的，從而控制LRRK2功能和激酶活性[24]。有趣的是，迄今為止描述的主要的致病性突變發生在LRRK2的酶促核心內，表明LRRK2活性的修飾極大地影響了PD的發病和進展。LRRK2 is a multidomain protein whose core contains two enzymatic functions. The GTPase domain consisting of the Ras of the complex protein (ROC) of the spacer domain terminating the C-terminus (COR) terminating the Roc domain is followed by the kinase domain belonging to the serine/threonine kinase. This enzymatic core is surrounded by a protein-protein interaction domain comprising a prion protein, an ankyrin, and a leucine-rich repeat (LRR) domain at the N-terminus of LRRK2 [23]. The C-terminus of LRRK2 contains the WD40 domain, which is thought to be required for protein folding, thereby controlling LRRK2 function and kinase activity [24]. Interestingly, the major pathogenic mutations described so far occur within the enzymatic core of LRRK2, suggesting that modification of LRRK2 activity greatly affects the onset and progression of PD.
迄今為止，近40種單氨基酸取代突變與常染色體顯性PD有關[25,26]。占歐洲家族性PD約6%以及占散發性PD病例3%的LRRK2最常見的突變形式包括Gly2019對Ser殘基的氨基酸取代。Gly2019位於保守的DYG-Mg2+ -結合基序內，在激酶結構域的子結構域-VII中[25]。最近的報導表明，這種突變增強了LRRK2的自磷酸化，以及其將髓磷脂鹼性蛋白磷酸化2-3倍的能力[8,12,27]。當在細胞系和原代神經元培養物中過表達G2019S-LRRK2時，觀察到在氧化應激不存在和存在下的細胞毒性，以及包涵體的形成[27,28]。這些結果以及LRRK2激酶活性的遺傳滅活顯示出對這種毒性表型的保護作用的事實，表明LRRK2激酶活性的改變潛在地參與LRRK2-PD的神經毒性和致病機制。To date, nearly 40 single amino acid substitution mutations have been associated with autosomal dominant PD [25,26]. The most common mutated forms of LRRK2, which account for about 6% of European familial PD and 3% of sporadic PD cases, include amino acid substitutions of Gly2019 for Ser residues. Gly2019 is located within the conserved DYG-Mg 2+ -binding motif, in the subdomain of the kinase domain -VII [25]. Recent reports indicate that this mutation enhances the autophosphorylation of LRRK2 and its ability to phosphorylate myelin basic protein by a factor of 2-3 [8,12,27]. When G2019S-LRRK2 was overexpressed in cell lines and primary neuronal cultures, cytotoxicity in the absence and presence of oxidative stress, and formation of inclusion bodies were observed [27, 28]. These results, along with the fact that genetic inactivation of LRRK2 kinase activity shows protection against this toxic phenotype, suggest that alterations in LRRK2 kinase activity are potentially involved in the neurotoxicity and pathogenic mechanisms of LRRK2-PD.
已經發現衍生自LRRK2 G2019S帕金森氏病患者的誘導多能幹細胞（iPSC）表現出神經突增生和對魚藤酮敏感性增加的缺陷，其可以通過遺傳校正G2019S突變或用具有LRRK2激酶活性的小分子抑制劑治療細胞加以改善[29]。與iPSC中的LRRK2 G2019S突變相關的增加的線粒體損傷也由G2019S突變的遺傳校正阻斷[30]。Induced pluripotent stem cells (iPSCs) derived from patients with LRRK2 G2019S Parkinson's disease have been found to exhibit neurite outgrowth and increased sensitivity to rotenone, which can be genetically corrected for G2019S mutations or by small molecules with LRRK2 kinase activity. Therapeutic cells are improved [29]. Increased mitochondrial damage associated with LRRK2 G2019S mutations in iPSCs is also blocked by genetic correction of the G2019S mutation [30].
另外的證據將LRRK2功能和功能障礙與自噬溶酶體途徑關聯[31]。LRRK2蛋白質導致伴侶介導的自噬中的缺陷，其對細胞降解α-突觸核蛋白的能力產生負面影響[32]。在其它細胞模型中，選擇性LRRK2抑制劑已被證明可以刺激巨噬細胞吞噬[33]。這些資料表明，具有LRRK2激酶活性的小分子抑制劑可以有效治療以由異常自噬/溶酶體降解途徑引起的細胞蛋白水解缺陷為特徵的疾病，包括與GBA突變相關的帕金森氏病[34]、其它α-突觸核蛋白病、tau蛋白病、阿爾茨海默病[35]和其它神經退行性疾病[36]和戈謝病[37]等形式。此外，與正常受試者的成纖維細胞相比，在C型Niemann-Pick（NPC）病患者的成纖維細胞中也觀察到LRRK2 mRNA的水準顯著升高，這表明異常的LRRK2功能可能在溶酶體疾病中起作用[38]。這一觀察結果表明LRRK2抑制劑可能有效治療NPC。Additional evidence links LRRK2 function and dysfunction to the autophagic lysosomal pathway [31]. LRRK2 protein causes a defect in chaperone-mediated autophagy that negatively affects the ability of cells to degrade alpha-synuclein [32]. In other cell models, selective LRRK2 inhibitors have been shown to stimulate macrophage phagocytosis [33]. These data indicate that small molecule inhibitors with LRRK2 kinase activity are effective in the treatment of diseases characterized by defective cell proteolysis caused by abnormal autophagy/lysosomal degradation pathways, including Parkinson's disease associated with GBA mutations [34] ], other forms of α-synuclein, tau, Alzheimer's disease [35] and other neurodegenerative diseases [36] and Gaucher disease [37]. In addition, a significant increase in the level of LRRK2 mRNA was observed in fibroblasts of patients with type C Niemann-Pick (NPC) disease compared to fibroblasts from normal subjects, suggesting that abnormal LRRK2 function may be soluble. It plays a role in enzymatic diseases [38]. This observation suggests that LRRK2 inhibitors may be effective in the treatment of NPC.
還報導了與PD相關的G2019S突變體形式的LRRK2增強微管蛋白相關性Tau的磷酸化[39]，並且已經提出了其中LRRK2作用於Tau和α-突觸核蛋白的致病作用上游的疾病模型[40]。為了支援這點，轉基因小鼠模型中LRRK2表達與增加的不溶性Tau聚集和增加的Tau磷酸化有關[41]。據報導，PD致病性突變體蛋白LRRK2 R1441 G的過度表達導致轉基因小鼠模型中帕金森氏病的症狀和Tau過度磷酸化[42]。因此，這些資料表明，具有激酶催化活性的LRRK2抑制劑可用於治療特徵為Tau過度磷酸化的tau蛋白病，例如嗜銀顆粒癡呆、皮克病、皮質基底核退化症、進行性核上性麻痹以及與染色體17相關的遺傳性額顳葉癡呆和帕金森氏綜合徵（FTDP-17）[43]。此外，LRRK2抑制劑可以具有治療特徵為多巴胺水準降低的其它疾病的效用，例如與藥物成癮相關的脫癮症狀/復發[44]。The PD20-associated G2019S mutant form of LRRK2 has also been shown to enhance the phosphorylation of tubulin-associated Tau [39], and diseases in which LRRK2 acts upstream of the pathogenic effects of Tau and α-synuclein have been proposed. Model [40]. To support this, LRRK2 expression in transgenic mouse models is associated with increased insoluble Tau aggregation and increased Tau phosphorylation [41]. Overexpression of the PD pathogenic mutant protein LRRK2 R1441 G has been reported to cause symptoms of Parkinson's disease and Tau hyperphosphorylation in a transgenic mouse model [42]. Therefore, these data indicate that kinase-catalyzed LRRK2 inhibitors can be used to treat tauopathy characterized by Tau hyperphosphorylation, such as argyrophilic dementia, Pico disease, cortical basal ganglia degeneration, progressive supranuclear palsy And hereditary frontotemporal dementia and Parkinson's syndrome (FTDP-17) associated with chromosome 17 [43]. In addition, LRRK2 inhibitors may have utility in treating other diseases characterized by decreased levels of dopamine, such as withdrawal symptoms/relapse associated with drug addiction [44].
在一方面，本文提供的是通式I的化合物或其立體異構體、互變異構體、N-氧化物、水合物、溶劑合物、代謝物、藥學上可接受的鹽、酯或前藥：
在一些實施方式中，R1 是H、C1-6 烷基、C3-7 環烷基、C3 -7 雜環烷基、C6 - 10 芳基、C3 -8 雜芳基、C1-3 烷基-C1 -7 雜芳基或C1-3 烷基-C6- 10 芳基，其中該C1-6 烷基、C3-7 環烷基、C3-7 雜環烷基、C6-10 芳基、C3 -8 雜芳基、C1-3 烷基-C1 -7 雜芳基，和C1-3 烷基-C6- 10 芳基各自獨立地且任選地被選自F、Cl、Br、I、-NO2 、-CN、-N3 、-NH2 、-OH、C1-6 烷基、C3-7 環烷基，和C3-7 雜環烷基中的一個以上的取代基取代。In some embodiments, R 1 is is H, C 1-6 alkyl, C 3-7 cycloalkyl, C 3 -7 heterocycloalkyl, C 6 - 10 aryl, C 3 -8 heteroaryl, -C 1 -7 C 1-3 alkyl or heteroaryl C 1-3 alkyl -C 6- 10 aryl group, wherein the C 1-6 alkyl, C 3-7 cycloalkyl, C 3-7 heterocycloalkyl, C 6-10 aryl, C 3 -8 heteroaryl, -C 1 -7 C 1-3 alkyl aryl, heteroaryl, C 1-3 alkyl, and aryl groups are each -C 6- 10 Independently and optionally selected from the group consisting of F, Cl, Br, I, -NO 2 , -CN, -N 3 , -NH 2 , -OH, C 1-6 alkyl, C 3-7 cycloalkyl, And substituted with one or more substituents in the C 3-7 heterocycloalkyl group.
在一些實施方式中，X1 是CO、-CH2 -、-(CH2 )2 -，或-(CH2 )3 -。In some embodiments, X 1 is CO, —CH 2 —, —(CH 2 ) 2 —, or —(CH 2 ) 3 —.
在一些實施方式中，Y是-CH2 -、-(CH2 )2 -、-(CH2 )3 -、-(CR2 R3 )-、C6 -10 芳基或C3 -8 雜芳基，任選地R2 和R3 與它們所連接的碳原子一起形成C3 -C8 碳環或3至8元雜環，其中該-CH2 -、-(CH2 )2 -，或-(CH2 )3 -、-(CR2 R3 )-、C6 -10 芳基、C3 -8 雜芳基、C3 -C8 碳環和3至8元雜環各自獨立地且任選地被選自F、Cl、Br、I、-NO2 、-CN、-N3 、-NH2 、-OH、C1-6 烷基，和C1-6 鹵代烷基中的一個以上的取代基取代。In some embodiments, Y is -CH 2 -, -(CH 2 ) 2 -, -(CH 2 ) 3 -, -(CR 2 R 3 )-, C 6 -10 aryl or C 3 -8 An aryl group, optionally R 2 and R 3 together with the carbon atom to which they are attached form a C 3 -C 8 carbocyclic ring or a 3 to 8 membered heterocyclic ring wherein -CH 2 -, -(CH 2 ) 2 -, Or -(CH 2 ) 3 -, -(CR 2 R 3 )-, C 6 -10 aryl, C 3 -8 heteroaryl, C 3 -C 8 carbocyclic and 3 to 8 membered heterocyclic rings are each independently And optionally one selected from the group consisting of F, Cl, Br, I, -NO 2 , -CN, -N 3 , -NH 2 , -OH, C 1-6 alkyl, and C 1-6 haloalkyl The above substituents are substituted.
在一些實施方式中，Y是-CH2
-、-(CH2
)2
-、-(CH2
)3
-、-(CR2
R3
)-，
在一些實施方式中，Z是鍵、NR2 、-CH2 -、-(CH2 )2 -、-(CH2 )3 -或-(CR2 R3 )-，其中該NR2 、-CH2 -、-(CH2 )2 -、-(CH2 )3 -和-(CR2 R3 )-各自獨立地且任選地被選自F、Cl、Br、I、-NO2 、-CN、-N3 、-NH2 、-OH、C1-6 烷基，和C1-6 鹵代烷基中的一個以上的取代基取代。In some embodiments, Z is a bond, NR 2 , —CH 2 —, —(CH 2 ) 2 —, —(CH 2 ) 3 —, or —(CR 2 R 3 )—, wherein the NR 2 , —CH 2 -, -(CH 2 ) 2 -, -(CH 2 ) 3 - and -(CR 2 R 3 )- are each independently and optionally selected from the group consisting of F, Cl, Br, I, -NO 2 , - One or more substituents of CN, -N 3 , -NH 2 , -OH, C 1-6 alkyl, and C 1-6 haloalkyl are substituted.
在一些實施方式中，R2 和R3 獨立地選自-H、C1-3 -烷基、C3-5 環烷基、C3-5 雜環烷基、C6-8 芳基或C3-8 雜芳基，其中該C1-3 烷基、C3-5 環烷基、C3-5 雜環烷基、C6-8 芳基和C3-8 雜芳基各自任選地且獨立地被選自F、Cl、Br、I、-NO2 、-CN、-N3 、-NH2 、-OH，和-CO2 H中的一個以上的取代基取代。In some embodiments, R 2 and R 3 are, independently, selected from -H, C 1-3 -alkyl, C 3-5 cycloalkyl, C 3-5 heterocycloalkyl, C 6-8 aryl or a C 3-8 heteroaryl group, wherein the C 1-3 alkyl group, the C 3-5 cycloalkyl group, the C 3-5 heterocycloalkyl group, the C 6-8 aryl group, and the C 3-8 heteroaryl group are each Optionally and independently substituted with one or more substituents selected from the group consisting of F, Cl, Br, I, -NO 2 , -CN, -N 3 , -NH 2 , -OH, and -CO 2 H.
在一些實施方式中，R1
選自以下基團：
在一些實施方式中，R1
較佳地選自以下基團：
在一些實施方式中，Y是-CH2 -、-(CH2 )2 -、-(CH2 )3 -、-(CR2 R3 )-、苯環、5至6元雜芳環、C6 - 10 芳基或C3 -8 雜芳基，任選地R2 和R3 與它們所連接的碳原子一起形成C3 -C6 碳環或3至6元雜環，其中該苯環、5至6元雜芳環、C6 - 10 芳基、C3 -8 雜芳基、C3 -C6 碳環和3至6元雜環各自獨立地且任選地被選自F、Cl、Br、I、-NO2 、-CN、-N3 、-NH2 、-OH、甲基、乙基、正丙基、異丙基、-CF3 ，和C1-3 鹵代烷基中的一個以上的取代基取代。In some embodiments, Y is -CH 2 -, -(CH 2 ) 2 -, -(CH 2 ) 3 -, -(CR 2 R 3 )-, benzene ring, 5 to 6 membered heteroaryl ring, C 6 - 10 aryl or C 3 -8 heteroaryl, optionally R 2 and R 3 together with the carbon atom to which they are attached form a C 3 -C 6 carbocyclic ring or a 3 to 6 membered heterocyclic ring, wherein the phenyl ring 5 to 6-membered heteroaryl ring, C 6 - 10 aryl, C 3 -8 heteroaryl, C 3 -C 6 carbocyclic ring, and 3 to six-independently and optionally substituted selected from F, Cl, Br, I, -NO 2 , -CN, -N 3 , -NH 2 , -OH, methyl, ethyl, n-propyl, isopropyl, -CF 3 , and C 1-3 haloalkyl Replaced by more than one substituent.
在一些實施方式中，Y選自以下基團：
在一些實施方式中，Y較佳地選自以下基團：
在一些實施方式中，本文提供了具有以下結構之一的化合物，或其立體異構體、互變異構體、N-氧化物、水合物、溶劑合物、代謝物、藥學上可接受的鹽、酯或前藥：
在一些實施方式中，本文提供了較佳地具有以下結構之一的化合物，或其立體異構體、互變異構體、N-氧化物、水合物、溶劑合物、代謝物、藥學上可接受的鹽、酯或前藥：
在另一方面，本文提供了包含本文揭露的化合物的藥物組合物。In another aspect, provided herein is a pharmaceutical composition comprising a compound disclosed herein.
在一些實施方式中，本文揭露的藥物組合物還包含藥學上可接受的載體、稀釋劑、輔料或其組合。In some embodiments, the pharmaceutical compositions disclosed herein further comprise a pharmaceutically acceptable carrier, diluent, adjuvant, or a combination thereof.
在一些實施方式中，本文揭露的藥物組合物還包含第二治療劑。In some embodiments, the pharmaceutical compositions disclosed herein further comprise a second therapeutic agent.
在另一方面，本文提供了本文揭露的化合物或藥物組合物在製備藥物中的用途，該藥物用於治療由癌症和神經退行性疾病中的至少一種引起的病症。In another aspect, provided herein is the use of a compound or pharmaceutical composition disclosed herein in the manufacture of a medicament for treating a condition caused by at least one of cancer and a neurodegenerative disease.
在一些實施方式中，神經退行性疾病是帕金森氏病。In some embodiments, the neurodegenerative disease is Parkinson's disease.
在另一方面，本文提供了治療由癌症和神經退行性疾病中的至少一種引起的病症的方法，包括給予受試者治療有效量的本文揭露的化合物或藥物組合物。In another aspect, provided herein is a method of treating a condition caused by at least one of cancer and a neurodegenerative disease, comprising administering to the subject a therapeutically effective amount of a compound or pharmaceutical composition disclosed herein.
在一些實施方式中，神經退行性疾病是帕金森氏病。In some embodiments, the neurodegenerative disease is Parkinson's disease.
在另一方面，本文提供了用於在治療由癌症和神經退行性疾病中的至少一種引起的病症中使用的的本文揭露的化合物或藥物組合物。In another aspect, provided herein are compounds or pharmaceutical compositions disclosed herein for use in treating a condition caused by at least one of cancer and a neurodegenerative disease.
在一些實施方式中，神經退行性疾病是帕金森氏病。In some embodiments, the neurodegenerative disease is Parkinson's disease.
在另一方面，本文提供了本文揭露的化合物或藥物組合物在製備藥物中的用途，該藥物用於預防或治療由任何異常激酶活性引起的、與之相關或伴隨的病症。In another aspect, provided herein is the use of a compound or pharmaceutical composition disclosed herein in the manufacture of a medicament for the prevention or treatment of a condition associated with or associated with any abnormal kinase activity.
在一些實施方式中，激酶是LRRK。In some embodiments, the kinase is LRRK.
在一些實施方式中，激酶是LRRK2。In some embodiments, the kinase is LRRK2.
在另一方面，本文提供了預防或治療由任何異常激酶活性引起的、與之相關或伴隨的病症的方法，包括給予受試者治療有效量的本文揭露的化合物或藥物組合物。In another aspect, provided herein is a method of preventing or treating a condition associated with or associated with any abnormal kinase activity comprising administering to a subject a therapeutically effective amount of a compound or pharmaceutical composition disclosed herein.
在一些實施方式中，激酶是LRRK。In some embodiments, the kinase is LRRK.
在一些實施方式中，激酶是LRRK2。In some embodiments, the kinase is LRRK2.
在另一方面，本文提供了用於在預防或治療由任何異常激酶活性引起的、與之相關或伴隨的病症中使用的的本文揭露的化合物或藥物組合物。In another aspect, provided herein are compounds or pharmaceutical compositions disclosed herein for use in preventing or treating a condition associated with or associated with any abnormal kinase activity.
在一些實施方式中，激酶是LRRK。In some embodiments, the kinase is LRRK.
在一些實施方式中，激酶是LRRK2。In some embodiments, the kinase is LRRK2.
在另一方面，本文提供了本文揭露的化合物或藥物組合物在試驗中的用途，該試驗用於鑒定能夠抑制激酶的其它候選化合物。In another aspect, provided herein is the use of a compound or pharmaceutical composition disclosed herein in an assay for identifying other candidate compounds capable of inhibiting a kinase.
在一些實施方式中，激酶是LRRK。In some embodiments, the kinase is LRRK.
在一些實施方式中，激酶是LRRK2。In some embodiments, the kinase is LRRK2.
在另一方面，本文提供了通式II的化合物，或其立體異構體、互變異構體、N-氧化物、水合物、溶劑合物、代謝物、藥學上可接受的鹽、酯或前藥：
在一些實施方式中，X1 是CO、-CH2 -、-(CH2 )2 -或-(CH2 )3 -。In some embodiments, X 1 is CO, —CH 2 —, —(CH 2 ) 2 —, or —(CH 2 ) 3 —.
在一些實施方式中，Y是-CH2 -、-(CH2 )2 -或-(CH2 )3 -。In some embodiments, Y is -CH 2 -, - (CH 2 ) 2 - or - (CH 2) 3 -.
在一些實施方式中，Z是鍵、N、-CH2 -、-(CH2 )2 -或-(CH2 )3 -。In some embodiments, Z is a bond, N, -CH 2 -, -(CH 2 ) 2 - or -(CH 2 ) 3 -.
在一些實施方式中，R4 各自獨立地是不存在的、F、Cl、Br、I、-NO2 、-CN、-N3 、-NH2 、-OH、C1-3 烷基或C1-3 鹵代烷基，任選地兩個連接至Y的R4 與Y一起形成C3 -C7 碳環或3至7元雜環，其中該C3 -C7 碳環和3至7元雜環各自獨立地且任選地被選自F、Cl、Br、I、-NO2 、-CN、-N3 、-NH2 、-OH、C1-6 烷基，和C1-6 鹵代烷基中的一個以上的取代基取代。In some embodiments, each R 4 is independently absent, F, Cl, Br, I, -NO 2 , -CN, -N 3 , -NH 2 , -OH, C 1-3 alkyl or C 1-3 haloalkyl, optionally two R 4 attached to Y together with Y form a C 3 -C 7 carbocyclic ring or a 3 to 7 membered heterocyclic ring wherein the C 3 -C 7 carbocyclic ring and 3 to 7 membered The heterocycles are each independently and optionally selected from the group consisting of F, Cl, Br, I, -NO 2 , -CN, -N 3 , -NH 2 , -OH, C 1-6 alkyl, and C 1-6 More than one substituent in the haloalkyl group is substituted.
在一些實施方式中，R5 是不存在的、F、Cl、Br、I、-NO2 、-CN、-N3 、-NH2 、-OH、C1-3 烷基或C1-6 鹵代烷基，任選地R4 和R5 與它們所連接的Y-Z一起形成苯環、C3 -C7 碳環、3至7元雜環或5至7元雜芳環，其中該苯環、C3 -C7 碳環、3至7元雜環和5至7元雜芳環各自獨立地且任選地被選自F、Cl、Br、I、-NO2 、-CN、-N3 、-NH2 、-OH、C1-6 烷基、C1-6 鹵代烷基中的一個以上的取代基取代。In some embodiments, R 5 is absent, F, Cl, Br, I, -NO 2 , -CN, -N 3 , -NH 2 , -OH, C 1-3 alkyl or C 1-6 a haloalkyl group, optionally R 4 and R 5 together with the YZ to which they are attached form a benzene ring, a C 3 -C 7 carbocyclic ring, a 3 to 7 membered heterocyclic ring or a 5 to 7 membered heteroaryl ring, wherein the phenyl ring, The C 3 -C 7 carbocyclic ring, the 3 to 7 membered heterocyclic ring and the 5 to 7 membered heteroaryl ring are each independently and optionally selected from the group consisting of F, Cl, Br, I, -NO 2 , -CN, -N 3 And one or more substituents of -NH 2 , -OH, C 1-6 alkyl, and C 1-6 haloalkyl are substituted.
在一些實施方式中，R1
選自以下基團：
在一些實施方式中，R5 是不存在的、F、Cl、Br、I、-NO2 、-CN、-N3 、-NH2 、-OH、C1-3 烷基或C1-3 鹵代烷基，任選地R4 和R5 與它們所連接的Y-Z一起形成苯環或吡唑環，其中該苯環和吡唑環各自獨立地且任選地被選自F、Cl、Br、-CN、-OH、-CO2 H，和-CF3 中的一個以上的取代基取代。In some embodiments, R 5 is absent, F, Cl, Br, I, -NO 2 , -CN, -N 3 , -NH 2 , -OH, C 1-3 alkyl or C 1-3 Haloalkyl, optionally R 4 and R 5 together with the YZ to which they are attached form a phenyl or pyrazole ring, wherein the phenyl ring and the pyrazole ring are each independently and optionally selected from the group consisting of F, Cl, Br, -CN, -OH, -CO 2 H, and one or more substituents in -CF 3 are substituted.
在另一方面，本文提供了包含本文揭露的通式II化合物的藥物組合物。In another aspect, provided herein is a pharmaceutical composition comprising a compound of formula II as disclosed herein.
在一些實施方式中，本文揭露的藥物組合物還包含藥學上可接受的載體、稀釋劑、輔料或其組合。In some embodiments, the pharmaceutical compositions disclosed herein further comprise a pharmaceutically acceptable carrier, diluent, adjuvant, or a combination thereof.
在一些實施方式中，本文揭露的藥物組合物還包含第二治療劑。In some embodiments, the pharmaceutical compositions disclosed herein further comprise a second therapeutic agent.
在另一方面，本文提供了通式II化合物或包含本文揭露的通式II化合物的藥物組合物在製備藥物中的用途，該藥物用於治療由癌症和神經退行性疾病中的至少一種引起的病症。In another aspect, provided herein is the use of a pharmaceutical composition of Formula II or a pharmaceutical composition comprising a compound of Formula II disclosed herein for the manufacture of a medicament for the treatment of at least one of cancer and a neurodegenerative disease. Illness.
在一些實施方式中，神經退行性疾病是帕金森氏病。In some embodiments, the neurodegenerative disease is Parkinson's disease.
在另一方面，本文提供了治療由癌症和神經退行性疾病中的至少一種引起的疾病的方法，包括向受試者給予治療有效量的通式II化合物或包含本文揭露的通式II化合物的藥物組合物。In another aspect, provided herein is a method of treating a condition caused by at least one of cancer and a neurodegenerative disease, comprising administering to a subject a therapeutically effective amount of a compound of Formula II or a compound of Formula II disclosed herein. Pharmaceutical composition.
在一些實施方式中，神經退行性疾病是帕金森氏病。In some embodiments, the neurodegenerative disease is Parkinson's disease.
在另一方面，本文提供了通式II化合物或包含本文揭露的通式II化合物的藥物組合物，其用於在治療由癌症和神經退行性疾病中的至少一種引起的病症中使用。In another aspect, provided herein is a compound of Formula II or a pharmaceutical composition comprising a compound of Formula II disclosed herein for use in the treatment of a condition caused by at least one of cancer and a neurodegenerative disease.
在一些實施方式中，神經退行性疾病是帕金森氏病。In some embodiments, the neurodegenerative disease is Parkinson's disease.
在另一方面，本文提供了通式II化合物或包含本文揭露的通式II化合物的藥物組合物在製備藥物中的用途，該藥物用於預防或治療由任何異常激酶活性引起的、與之相關或伴隨的病症。In another aspect, provided herein is the use of a pharmaceutical composition of Formula II or a pharmaceutical composition comprising a compound of Formula II disclosed herein for the manufacture of a medicament for use in preventing or treating a disease caused by any abnormal kinase activity Or accompanying illness.
在一些實施方式中，激酶是LRRK。In some embodiments, the kinase is LRRK.
在一些實施方式中，激酶是LRRK2。In some embodiments, the kinase is LRRK2.
在另一方面，本文提供了預防或治療由任何異常激酶活性引起的、與之相關或伴隨的病症的方法，包括向受試者給予治療有效量的通式II化合物或包含本文揭露的通式II化合物的藥物組合物。In another aspect, provided herein is a method of preventing or treating a condition associated with or associated with any abnormal kinase activity comprising administering to a subject a therapeutically effective amount of a compound of Formula II or a formula disclosed herein. A pharmaceutical composition of a compound of II.
在一些實施方式中，激酶是LRRK。In some embodiments, the kinase is LRRK.
在一些實施方式中，激酶是LRRK2。In some embodiments, the kinase is LRRK2.
在另一方面，本文提供了通式II化合物或包含本文揭露的通式II化合物的藥物組合物，其用於在預防或治療由任何異常激酶活性引起的、與之相關或伴隨的病症中使用。In another aspect, provided herein is a compound of Formula II or a pharmaceutical composition comprising a compound of Formula II disclosed herein for use in preventing or treating a condition associated with or associated with any abnormal kinase activity .
在一些實施方式中，激酶是LRRK。In some embodiments, the kinase is LRRK.
在一些實施方式中，激酶是LRRK2。In some embodiments, the kinase is LRRK2.
在另一方面，本文提供了通式II化合物或包含本文揭露的通式II化合物的藥物組合物在試驗中的用途，該試驗用於鑒定能夠抑制激酶的另外候選化合物。In another aspect, provided herein is the use of a pharmaceutical composition of Formula II or a pharmaceutical composition comprising a compound of Formula II disclosed herein for use in the identification of additional candidate compounds capable of inhibiting kinase.
在一些實施方式中，激酶是LRRK。In some embodiments, the kinase is LRRK.
在一些實施方式中，激酶是LRRK2。In some embodiments, the kinase is LRRK2.
定義和常規術語 本發明中引用的所有參考文獻通過引用將其整體併入本文，並且在引入的參考文獻和本發明之間存在不一致的情況下，將以本揭露為準。此外，本文使用的所有術語和短語具有本領域技術人員已知的一般含義。即使如此，仍然需要對本發明的術語和短語進行更詳細的說明。在提及的術語和短語與熟知的意義之間存在不一致的情況下，以本揭露為準。不管所討論的術語是單獨地還是以組合出現，本說明書中使用的常規術語的以下定義均適用。 Definitions and General Terms All references cited in the present invention are hereby incorporated by reference in their entirety, and in the case of inconsistency between the incorporated references and the present invention. Moreover, all terms and phrases used herein have the ordinary meaning as known to those skilled in the art. Even so, the terms and phrases of the present invention need to be described in more detail. In the event of any inconsistency between the terms and phrases referred to and well-known meanings, the present disclosure will control. The following definitions of conventional terms used in this specification apply regardless of whether the terms in question appear individually or in combination.
如在本文中使用的語法冠詞“一個（a）”、“一種（an）”和“該（the）”旨在包括“至少一個”或“一個以上”，除非在本中另有說明或與上下文明顯矛盾。因此，本文所使用的冠詞是指一個或多於一個（即，至少一個）的該冠詞的語法物件。作為示例，“組分”是指一個以上的組分，並且因此可能考慮多於一個的組分，並且可以在所描述的實施方式的實現中採用或使用。The grammatical articles "a", "an", "the" and "the" The context is clearly contradictory. Thus, the article as used herein refers to one or more than one (ie, at least one) of the grammatical items of the article. By way of example, "component" refers to more than one component, and thus more than one component may be considered and may be employed or used in the practice of the described embodiments.
如在本文中所述的，本文揭露的化合物可以任選地被一個以上的取代基取代，如通常在以下舉例說明的，或由本發明的特定類別、亞類和種類舉例說明的。As described herein, the compounds disclosed herein may be optionally substituted with more than one substituent, as exemplified generally below, or by the particular classes, subclasses, and species of the invention.
術語“鹵素”是指氟（F）、氯（Cl）、溴（Br）或碘（I）。The term "halogen" means fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).
術語“烷基”是指1至10個碳原子的飽和直鏈或支鏈單價烴基。除非另有說明，烷基基團含有1-10個碳原子。在一些實施方式中，烷基基團含有1-8個碳原子；在其它實施方式中，烷基基團含有1-6個碳原子；在又一其它實施方式中，烷基基團含有1-4個碳原子；在再一其它實施方式中，烷基基團含有1-3個碳原子。烷基基團任選地被一個以上的本文所描述的取代基取代。The term "alkyl" refers to a saturated straight or branched chain monovalent hydrocarbon radical of from 1 to 10 carbon atoms. Unless otherwise stated, an alkyl group contains from 1 to 10 carbon atoms. In some embodiments, the alkyl group contains from 1 to 8 carbon atoms; in other embodiments, the alkyl group contains from 1 to 6 carbon atoms; in yet other embodiments, the alkyl group contains 1 - 4 carbon atoms; in still other embodiments, the alkyl group contains 1-3 carbon atoms. The alkyl group is optionally substituted with more than one substituent described herein.
烷基基團的一些非限制性實例包括甲基（Me，-CH3 ）、乙基（Et，-CH2 CH3 ）、正丙基（n -Pr，-CH2 CH2 CH3 ）、異丙基（i -Pr，-CH(CH3 )2 ）、正丁基（n -Bu，-CH2 CH2 CH2 CH3 ）、異丁基（i -Bu，-CH2 CH(CH3 )2 ）、仲丁基（s -Bu，-CH(CH3 )CH2 CH3 ）、叔丁基（t -Bu，-C(CH3 )3 ）、正戊基（-CH2 CH2 CH2 CH2 CH3 ）、2-戊基（-CH(CH3 )CH2 CH2 CH3 ）、3-戊基（-CH(CH2 CH3 )2 ）、正己基（-CH2 CH2 CH2 CH2 CH2 CH3 ）、2-己基（-CH(CH3 )CH2 CH2 CH2 CH3 ）、3-己基（-CH(CH2 CH3 )(CH2 CH2 CH3 )）等。Some non-limiting examples of alkyl groups include methyl (Me, -CH 3 ), ethyl (Et, -CH 2 CH 3 ), n-propyl ( n- Pr, -CH 2 CH 2 CH 3 ), Isopropyl ( i -Pr, -CH(CH 3 ) 2 ), n-butyl ( n -Bu, -CH 2 CH 2 CH 2 CH 3 ), isobutyl ( i -Bu, -CH 2 CH(CH) 3 ) 2 ), sec-butyl ( s- Bu, -CH(CH 3 )CH 2 CH 3 ), tert-butyl ( t -Bu, -C(CH 3 ) 3 ), n-pentyl (-CH 2 CH) 2 CH 2 CH 2 CH 3 ), 2-pentyl (-CH(CH 3 )CH 2 CH 2 CH 3 ), 3-pentyl (-CH(CH 2 CH 3 ) 2 ), n-hexyl (-CH 2 ) CH 2 CH 2 CH 2 CH 2 CH 3 ), 2-hexyl (-CH(CH 3 )CH 2 CH 2 CH 2 CH 3 ), 3-hexyl (-CH(CH 2 CH 3 )(CH 2 CH 2 CH 3 )) etc.
術語“環烷基”是指具有3至10個碳原子的單價或多價飽和環，如單環、雙環或三環系統。並且其中雙環或三環系統可以包括稠環、橋環和螺環。在一些實施方式中，環烷基基團含有3至8個碳原子。在其它實施方式中，環烷基基團含有3至6個碳原子。環烷基（cycloalkyl radical）任選被一個以上的本文所描述的取代基取代。The term "cycloalkyl" refers to a monovalent or polyvalent saturated ring having from 3 to 10 carbon atoms, such as a monocyclic, bicyclic or tricyclic system. And wherein the bicyclic or tricyclic system can include a fused ring, a bridged ring, and a spiro ring. In some embodiments, a cycloalkyl group contains from 3 to 8 carbon atoms. In other embodiments, the cycloalkyl group contains from 3 to 6 carbon atoms. The cycloalkyl radical is optionally substituted with more than one substituent described herein.
術語“芳基”是指具有總共6至12個環成員，較佳6至10個環成員，且更佳6個環成員的單價或多價單環、雙環或三環碳環系統，並且其中該系統中的至少一個環是芳香族的。芳基基團通常但不一定通過芳基基團的芳香族環與母體分子結合。術語“芳基”和“芳香環”在本文中可以互換使用。芳基基團的實例可以包括苯基、萘基、蒽等。芳基（aryl radical）任選被一個以上的本文所描述的取代基取代。The term "aryl" refers to a monovalent or multivalent monocyclic, bicyclic or tricyclic carbocyclic ring system having a total of from 6 to 12 ring members, preferably from 6 to 10 ring members, and more preferably 6 ring members, and wherein At least one ring in the system is aromatic. An aryl group typically, but not necessarily, binds to the parent molecule through an aromatic ring of an aryl group. The terms "aryl" and "aromatic ring" are used interchangeably herein. Examples of the aryl group may include a phenyl group, a naphthyl group, an anthracene or the like. The aryl radical is optionally substituted with more than one substituent described herein.
術語“雜芳基”是指具有總共5至10個環成員的單價或多價單環、雙環或三環系統，並且其中系統中的至少一個環是芳香族的，並且至少一個環含有一個以上的雜原子。雜芳基基團通常但不一定通過雜芳基基團的芳香環與母體分子結合。術語“雜芳基”和“雜芳香環”或“雜芳香化合物”在本文中可以互換使用。雜芳基基團任選被一個以上的本文揭露的取代基取代。在一些實施方式中，5至10元雜芳基基團含有1、2、3或4個獨立地選自O、S和N的雜原子；在一些其它實施方式中，5至6元雜芳基是單環系統並且含有1、2、3或4個獨立地選自O、S和N的雜原子。The term "heteroaryl" refers to a monovalent or multivalent monocyclic, bicyclic or tricyclic ring system having a total of from 5 to 10 ring members, and wherein at least one ring in the system is aromatic and at least one ring contains more than one Hetero atom. A heteroaryl group typically, but not necessarily, binds to the parent molecule through an aromatic ring of a heteroaryl group. The terms "heteroaryl" and "heteroaromatic ring" or "heteroaromatic compound" are used interchangeably herein. The heteroaryl group is optionally substituted with more than one substituent disclosed herein. In some embodiments, the 5 to 10 membered heteroaryl group contains 1, 2, 3 or 4 heteroatoms independently selected from O, S and N; in some other embodiments, 5 to 6 membered heteroaryl The base is a single ring system and contains 1, 2, 3 or 4 heteroatoms independently selected from O, S and N.
雜芳基環的一些非限制性實例包括噻吩基、呋喃基、吡咯基、吡啶基、噁唑基、噻唑基、咪唑基、吡唑基、異噁唑基、異噻唑基、噁二唑基、***基、噻二唑基、四唑基等及其苯並衍生物，如苯並呋喃基、苯並噻吩基、苯並咪唑基、吲哚基、異吲哚基、吲唑基等；或吡啶基、吡嗪基、嘧啶基、噠嗪基、三嗪基等及其苯並衍生物，如喹啉基、異喹啉基、噌啉基、酞嗪基、喹唑啉基、喹喔啉基、萘啶基等。Some non-limiting examples of heteroaryl rings include thienyl, furyl, pyrrolyl, pyridyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl , triazolyl, thiadiazolyl, tetrazolyl, etc. and benzo derivatives thereof, such as benzofuranyl, benzothienyl, benzimidazolyl, indolyl, isodecyl, carbazolyl, etc. Or pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl and the like and benzo derivatives thereof, such as quinolyl, isoquinolyl, porphyrinyl, pyridazinyl, quinazolinyl, Quinoxalinyl, naphthyridyl and the like.
“雜環烷基”是指含有一個以上的選自氮、氧和硫的雜原子的環狀脂肪族基團，在環中其任選地被一個以上的-(CO)-基團中斷和/或在環中任選地含有一個以上的雙鍵。可替換地，雜環烷基基團是C4-7 -雜環烷基、更佳C4-6 -雜環烷基。較佳的雜環烷基基團包括但不限於呱嗪基、呱啶基、嗎啉基、硫代嗎啉基、吡咯烷基、四氫呋喃基和四氫吡喃基。本發明化合物的描述 治療應用 "Heterocycloalkyl" means a cyclic aliphatic group containing one or more heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, which are optionally interrupted by more than one -(CO)- group in the ring. / or optionally containing more than one double bond in the ring. Alternatively, the heterocycloalkyl group is a C 4-7 -heterocycloalkyl group, more preferably a C 4-6 -heterocycloalkyl group. Preferred heterocycloalkyl groups include, but are not limited to, pyridazinyl, acridinyl, morpholinyl, thiomorpholinyl, pyrrolidinyl, tetrahydrofuranyl and tetrahydropyranyl. Description of the compounds of the invention for therapeutic applications
本發明的另一方面涉及上述化合物用於在醫藥中使用。Another aspect of the invention relates to the use of the above compounds for use in medicine.
本發明的另一方面涉及上述化合物用於在治療癌症或神經退行性疾病中使用。Another aspect of the invention relates to the use of the above compounds for the treatment of cancer or neurodegenerative diseases.
另一方面涉及如上述化合物在製備藥物中的用途，該藥物用於治療或預防神經退行性疾病。較佳地，神經退行性疾病是帕金森氏病。Another aspect relates to the use of a compound as described above for the preparation of a medicament for the treatment or prevention of a neurodegenerative disease. Preferably, the neurodegenerative disease is Parkinson's disease.
另一方面涉及上述化合物在製備藥物中的用途，該藥物用於治療或預防增殖性疾病，例如癌症。Another aspect relates to the use of a compound as described above for the manufacture of a medicament for the treatment or prevention of a proliferative disorder, such as cancer.
較佳地，以足以抑制一種以上的激酶、較佳LRRK、甚至更佳LRRK2的量給予該化合物。Preferably, the compound is administered in an amount sufficient to inhibit more than one kinase, preferably LRRK, even better LRRK2.
又一其它方面涉及本發明化合物在製備藥物中的用途，該藥物用於預防或治療由針對於生物靶標的任何異常活性引起的、與之相關或伴隨的病症，其中該靶標是激酶，更佳LRRK，甚至更佳LRRK2。Still another aspect relates to the use of a compound of the invention in the manufacture of a medicament for the prevention or treatment of a disorder associated with or associated with any abnormal activity against a biological target, wherein the target is a kinase, preferably LRRK, even better LRRK2.
較佳地，該病症是帕金森氏病。Preferably, the condition is Parkinson's disease.
本發明的另一方面涉及治療蛋白激酶相關疾病或病症的方法。根據本發明該方面的方法是通過將治療有效量的如上所述的本發明化合物本身，或更佳作為與例如如下文詳述的藥學上可接受的載體混合的部分藥物組合物給予至需要其的受試者而實現的。Another aspect of the invention relates to a method of treating a protein kinase related disease or condition. The method according to this aspect of the invention is by administering a therapeutically effective amount of a compound of the invention as described above per se, or better as part of a pharmaceutical composition, for example mixed with a pharmaceutically acceptable carrier as detailed below, to the desired The subject is achieved.
本發明的又另一方面涉及治療患有通過抑制蛋白激酶而減輕的疾病狀態的哺乳動物的方法，其中該方法包括向哺乳動物給予治療有效量的本發明化合物。Yet another aspect of the invention relates to a method of treating a mammal having a disease state attenuated by inhibition of a protein kinase, wherein the method comprises administering to the mammal a therapeutically effective amount of a compound of the invention.
較佳地，疾病狀態是通過抑制蛋白激酶LRRK，更佳LRRK2來減輕的。Preferably, the disease state is alleviated by inhibition of the protein kinase LRRK, more preferably LRRK2.
較佳地，哺乳動物是人類。Preferably, the mammal is a human.
術語“方法”是指用於完成給定任務的方式、手段、技術和程式，包括但不限於化學、藥理學、生物學、生物化學和醫學領域的從業者已知的或由他們從已知的方式、手段、技術和程式易於開發的那些方式、手段、技術和程式。The term "method" refers to the manner, means, techniques, and procedures used to accomplish a given task, including but not limited to those known to those skilled in the chemical, pharmacological, biological, biochemical, and medical fields. Ways, means, techniques, and programs that are easy to develop in ways, means, techniques, and programs.
如本文所使用的術語“給藥”是指將本發明化合物與蛋白激酶組合在一起的方法，以這種方式使得化合物可以影響蛋白激酶的酶活性，通過直接地，即通過與蛋白激酶本身相互作用或者間接地，即通過與在其上蛋白激酶的催化活性是依賴性的其它分子相互作用。如本文所使用的，可以在體外（即在試管中），或體內（即在活體的細胞或組織中）完成給藥。The term "administering" as used herein refers to a method of combining a compound of the invention with a protein kinase in such a way that the compound can affect the enzymatic activity of the protein kinase by directly, ie, by interacting with the protein kinase itself. The interaction or indirectly, ie by interaction with other molecules on which the catalytic activity of the protein kinase is dependent. As used herein, administration can be accomplished in vitro (i.e., in a test tube), or in vivo (i.e., in cells or tissues of a living organism).
在本文中，術語“治療”包括消除、基本上抑制、減緩或逆轉疾病或病症的進展，基本上改善疾病或病症的臨床症狀或基本上預防疾病或病症的臨床症狀出現。As used herein, the term "treating" includes eliminating, substantially inhibiting, slowing or reversing the progression of a disease or condition, substantially ameliorating the clinical symptoms of the disease or condition or substantially preventing the appearance of a clinical condition of the disease or condition.
在本文中，術語“預防”是指起初阻止生物體獲得病症或疾病的方法。As used herein, the term "prevention" refers to a method of initially preventing an organism from acquiring a condition or disease.
術語“治療有效量”是指所給予量的化合物將在一定程度上緩解被治療的疾病或病症的一種以上的症狀。The term "therapeutically effective amount" means that the administered amount of the compound will alleviate to a certain extent more than one symptom of the disease or condition being treated.
對於本發明中使用的任何化合物，治療有效量（本文中也稱為治療有效劑量）可以最初地由細胞培養測定來進行估計。例如，可以配製劑量以在動物模型中實現包括細胞培養中測定的IC50 或IC100 的迴圈濃度範圍。這些資訊可用於更精確地確定人體中的有用劑量。也可以從體內資料估計初始劑量。使用這些初步指導，本領域普通技術人員可以確定人類的有效劑量。For any compound used in the present invention, a therapeutically effective amount (also referred to herein as a therapeutically effective dose) can be estimated initially from cell culture assays. For example, a dose can be formulated to achieve a loop comprising IC 100 or IC 50 concentration range determined in cell culture in animal models. This information can be used to more accurately determine the useful dose in the human body. The initial dose can also be estimated from in vivo data. Using these preliminary guidelines, one of ordinary skill in the art can determine the effective dosage for humans.
此外，可以通過細胞培養物或實驗動物中的標準藥物程式來確定本文該化合物的毒性和治療功效，例如通過測定LD50 和ED50 。毒性和治療有效之間的劑量比是治療指標，並且可以表示為LD50 和ED50 之間的比率。表現出高治療指數的化合物是較佳的。由這些細胞培養物測定和動物研究所獲得的資料可用於配製在人體中使用而無毒性的劑量範圍。這些化合物的劑量較佳在包括具有很少或無毒性的ED50 的迴圈濃度的範圍內。劑量可以在該範圍內變化，這取決於所用的劑型和採用的給藥途徑。鑒於患者狀況，個體醫師可以選擇確切的製劑、給藥途徑和劑量。（參見，例如，Fingl等人，1975，In：The Pharmacological Basis of Therapeutics，第1章，第1頁）。Further, it is possible to determine the toxicity and therapeutic efficacy of the compounds described herein by standard pharmaceutical cell culture or experimental animals program, for example, by determining the LD 50 and ED 50. The dose ratio between toxic and therapeutic effective therapeutic index and may be expressed as the ratio between the 50 LD 50 and ED. Compounds which exhibit a high therapeutic index are preferred. The data obtained from these cell culture assays and animal studies can be used to formulate dosage ranges for use in humans without toxicity. The dosage of such compounds preferably in the range comprises loops with little or no toxicity concentration of ED 50. The dosage can vary within this range, depending on the dosage form employed and the route of administration employed. Given the patient's condition, the individual physician can choose the exact formulation, route of administration and dosage. (See, for example, Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Chapter 1, page 1).
可以單獨調整劑量和間隔以提供足以維持治療效果的活性化合物的血漿水準。用於口服給藥的常規患者劑量的範圍為約1-2000 mg/kg/天，通常為約2-1000 mg/kg/天，較佳為約5-700 mg/kg/天且最佳為約10-500 mg/kg/天。較佳地，通過每天給予多個劑量來實現治療有效的血清水準。在局部給藥或選擇性攝取的情況下，藥物的有效局部濃度可能與血漿濃度無關。本領域技術人員將能夠優化治療有效的局部劑量而無需過多的實驗。The dosages and intervals can be adjusted individually to provide a plasma level of the active compound sufficient to maintain the therapeutic effect. Conventional patient doses for oral administration range from about 1 to 2000 mg/kg/day, usually from about 2 to 1000 mg/kg/day, preferably from about 5 to 700 mg/kg/day, and most preferably About 10-500 mg/kg/day. Preferably, a therapeutically effective serum level is achieved by administering multiple doses per day. In the case of topical administration or selective uptake, the effective local concentration of the drug may be independent of plasma concentration. Those skilled in the art will be able to optimize therapeutically effective topical doses without undue experimentation.
如本文所使用的，“激酶相關的疾病或病症”是指以本文定義的不適當激酶活性或激酶的過度活性為特徵的疾病或病症。不適當活性是指（i）通常不表達該激酶的細胞中的激酶表達；（ii）增加的激酶表達，導致不希望的細胞增殖、分化和/或生長；或（iii）降低的激酶表達，導致細胞增殖、分化和/或生長不期望的減少。激酶的過度活性是指編碼特定激酶的基因的擴增或一定激酶活性水準的產生，其可以與細胞增殖、分化和/或生長紊亂相關（即，隨著激酶水準增加，一個以上的細胞紊亂的症狀的嚴重程度增加）。過度活性也可以是由於突變而導致的與配體無關或組成型啟動的結果，該突變如負責配體結合的激酶的片段缺失。As used herein, "kinase-associated disease or disorder" refers to a disease or condition characterized by inappropriate kinase activity as defined herein or overactivity of a kinase. Inappropriate activity refers to (i) kinase expression in cells that normally do not express the kinase; (ii) increased kinase expression leading to undesirable cell proliferation, differentiation and/or growth; or (iii) decreased kinase expression, Undesirable reduction in cell proliferation, differentiation and/or growth. Overactivity of a kinase refers to the amplification of a gene encoding a particular kinase or the production of a certain kinase activity level, which can be associated with cell proliferation, differentiation and/or growth disorders (ie, more than one cell disorder as the level of kinase increases) The severity of the symptoms increases). Excessive activity may also be the result of ligand-independent or constitutive initiation due to mutations, such as deletion of a fragment of a kinase responsible for ligand binding.
本文所述的化合物可用於預防的較佳疾病或病症包括癌症和神經退行性疾病，如帕金森氏病。Preferred diseases or conditions for which the compounds described herein are useful for prevention include cancer and neurodegenerative diseases such as Parkinson's disease.
因此，本發明還提供了本文定義的化合物用於製備藥物的用途，該藥物用於治療期望以抑制LRRK2的疾病。這些疾病包括帕金森氏病。藥物組合物 Accordingly, the invention also provides the use of a compound as defined herein for the manufacture of a medicament for the treatment of a disease which is desired to inhibit LRRK2. These diseases include Parkinson's disease. Pharmaceutical composition
對於根據本發明的用途，本文所述的化合物或其生理上可接受的鹽、酯或其它生理功能衍生物可以作為藥物製劑呈現，該藥物製劑包含化合物或其生理上可接受的鹽、酯或其它生理功能衍生物，以及一種以上的藥學上可接受的載體，以及任選的其它治療和/或預防成分。載體在與製劑的其它成分相容的意義上必須是可接受的並且對其接受者無害。藥物組合物可用於人和獸醫中的人或動物用途。For the use according to the invention, the compounds described herein, or physiologically acceptable salts, esters or other physiologically functional derivatives thereof, may be presented as pharmaceutical preparations comprising a compound or a physiologically acceptable salt, ester or Other physiologically functional derivatives, as well as more than one pharmaceutically acceptable carrier, and optionally other therapeutic and/or prophylactic ingredients. The carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The pharmaceutical compositions are useful for human or animal use in humans and veterinarians.
用於本文描述的各種不同形式的藥物組合物的合適輔料的實例可以在“Handbook of Pharmaceutical Excipients，第2版，（1994），由A Wade和PJ Weller編輯”中找到。Examples of suitable excipients for the various different forms of pharmaceutical compositions described herein can be found in "Handbook of Pharmaceutical Excipients, 2nd Edition, (1994), edited by A Wade and PJ Weller."
用於治療用途的可接受的載體或稀釋劑在製藥領域是熟知的，並且在例如Remington's Pharmaceutical Sciences，Mack Publishing Co.（A.R. Gennaro編輯1985）中進行了描述。Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro, ed. 1985).
合適載體的實例包括乳糖、澱粉、葡萄糖、甲基纖維素、硬脂酸鎂、甘露糖醇、山梨糖醇等。合適稀釋劑的實例包括乙醇、甘油和水。Examples of suitable carriers include lactose, starch, dextrose, methylcellulose, magnesium stearate, mannitol, sorbitol, and the like. Examples of suitable diluents include ethanol, glycerin and water.
藥物載體、輔料或稀釋劑的選擇可以根據預期給藥途徑和標準藥學實踐來進行選擇。藥物組合物可以包含或額外包含作為載體、輔料或稀釋劑的任何合適的黏合劑、潤滑劑、懸浮劑、包衣劑、增溶劑、緩衝劑、調味劑、表面活性劑、增稠劑、防腐劑（包括抗氧化劑）等，以及為了使得製劑與意圖的接受者的血液等滲所包含的物質。The choice of pharmaceutical carrier, adjuvant or diluent can be selected based on the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may comprise or additionally comprise any suitable binder, lubricant, suspending agent, coating agent, solubilizing agent, buffering agent, flavoring agent, surfactant, thickening agent, antiseptic as a carrier, adjuvant or diluent. The agent (including an antioxidant) or the like, and a substance contained in order to make the preparation isotonic with the blood of the intended recipient.
合適黏合劑的實例包括澱粉、明膠、天然糖（如葡萄糖、無水乳糖、游離乳糖、β-乳糖）、玉米甜味劑、天然和合成膠（如***膠、黃芪膠）或海藻酸鈉、羧甲基纖維素和聚乙二醇。Examples of suitable binders include starch, gelatin, natural sugars (such as glucose, anhydrous lactose, free lactose, beta-lactose), corn sweeteners, natural and synthetic gums (such as acacia, tragacanth) or sodium alginate, carboxy. Methyl cellulose and polyethylene glycol.
合適潤滑劑的實例包括油酸鈉、硬脂酸鈉、硬脂酸鎂、苯甲酸鈉、乙酸鈉、氯化鈉等。Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like.
可以在藥物組合物中提供防腐劑、穩定劑、染料甚至調味劑。防腐劑的實例包括苯甲酸鈉、山梨酸和對羥基苯甲酸的酯。也可以使用抗氧化劑和懸浮劑。Preservatives, stabilizers, dyes and even flavoring agents can be provided in the pharmaceutical compositions. Examples of preservatives include esters of sodium benzoate, sorbic acid, and p-hydroxybenzoic acid. Antioxidants and suspending agents can also be used.
藥物製劑包括適合於口服、局部（包括皮膚、口腔和舌下）、直腸或腸胃外（包括皮下、皮內、肌內和靜脈內）、鼻內和肺部給藥（例如，通過吸入）的那些。製劑可以在適當的情況下以離散劑量單位方便地呈現，並且可以通過藥學領域眾所周知的任何方法進行製備。所有方法包括讓活性化合物與液體載體或細碎固體載體或兩者聯合的步驟，然後如果需要的話，將產物成形為所需製劑。Pharmaceutical formulations include those suitable for oral, topical (including dermal, buccal and sublingual), rectal or parenteral (including subcutaneous, intradermal, intramuscular, and intravenous), intranasal, and pulmonary administration (eg, by inhalation). Those ones. The formulations may conveniently be presented in discrete dosage units where appropriate and may be prepared by any methods known in the art of pharmacy. All methods include the step of bringing the active compound into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation.
其中載體是固體的適用於口服給藥的藥物製劑最佳地以單位劑量製劑形式，如各自含有預定量的活性化合物的丸劑、膠囊或片劑。可以通過壓製或模製，任選地與一種以上的輔助成分一起製備片劑。可以通過在合適的機器中壓製處於自由流動形式（諸如粉末或顆粒）的活性化合物，任選地與黏合劑、潤滑劑、惰性稀釋劑、潤滑物質、表面活性劑或分散劑混合來製備壓製片劑。可以通過模製活性化合物和惰性液體稀釋劑來製備模製的片劑。可以任選地將片劑包衣，如果不進行包衣的話，可以任選地列印符號。可以通過將活性化合物單獨地或與一種以上的輔助成分混合填充到膠囊殼中，然後以常規方式進行密封來製備膠囊。扁囊劑類似於膠囊，其中將活性化合物與任何輔助成分一起密封在米紙膜中。也可以將活性化合物配製成可分散的顆粒，例如可以在給藥前將其懸浮於水中，或灑在食物上。可以將顆粒包裝在例如小袋中。其中載體是液體的適合於口服給藥的製劑，可以作為以水性或非水性液體方式的溶液或懸浮液，或作為水包油液體乳劑呈現。The pharmaceutical preparations suitable for oral administration wherein the carrier is solid are preferably in the form of unit dosage formulations, such as pills, capsules or tablets each containing a predetermined amount of active compound. Tablets may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing the active compound in a free-flowing form, such as a powder or granule, in a suitable machine, optionally with a binder, lubricant, inert diluent, lubricant, surfactant or dispersion. Agent. Molded tablets can be prepared by molding the active compound and inert liquid diluent. The tablets may optionally be coated, and if not coated, the symbols may optionally be printed. Capsules can be prepared by filling the active compound into the capsule shell, either alone or in combination with one or more accessory ingredients, and then sealing in a conventional manner. The cachet is similar to a capsule in which the active compound is enclosed in a rice paper film together with any auxiliary ingredients. The active compound can also be formulated as a dispersible granule, for example, it can be suspended in water or sprinkled on the food before administration. The granules can be packaged, for example, in a sachet. Formulations suitable for oral administration wherein the carrier is a liquid may be presented as a solution or suspension in the form of an aqueous or nonaqueous liquid, or as an oil-in-water emulsion.
用於口服給藥的製劑包括控釋劑型，例如片劑，其中將活性化合物配製於合適的控釋基質中，或將其包衣有合適的釋控膜。這類製劑可以特別方便地用於預防性使用。Formulations for oral administration include controlled release dosage forms such as tablets, wherein the active compound is formulated in a suitable controlled release medium or coated with a suitable release film. Such formulations are particularly convenient for prophylactic use.
適用於直腸給藥的藥物製劑（其中載體是固體）最佳地以單位劑量栓劑形式呈現。合適的載體包括可可脂和本領域通常使用的其它材料。可以通過將活性化合物與軟化或熔化的載體混合，然後在模具中冷卻和成型來方便地形成栓劑。適用於腸胃外給藥的藥物製劑包括在水性或油性溶劑中的活性化合物的無菌溶液或懸浮液。Pharmaceutical formulations suitable for rectal administration wherein the carrier is a solid are optimally presented as unit dosage suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppository can be conveniently formed by mixing the active compound with a softened or melted carrier, followed by cooling and shaping in a mold. Pharmaceutical preparations suitable for parenteral administration include sterile solutions or suspensions of active compounds in aqueous or oily solvents.
可注射的製劑可適用於彈丸注射（bolus injection）或連續注射。這種製劑方便地存在於單位劑量或多劑量容器中，在引入製劑後將容器密封直到使用需要。可替換地，活性化合物可以是粉末形式，在使用前由合適的溶劑，如無菌的無熱原水形成。Injectable formulations are suitable for bolus injection or continuous injection. Such formulations are conveniently presented in unit or multi-dose containers, which are sealed after introduction into the formulation until use. Alternatively, the active compound may be in powder form, formed by a suitable solvent, such as sterile pyrogen-free water, before use.
也可以將活性化合物配製成長效持久製劑，其可以通過肌內注射或通過植入，例如皮下或肌肉內給藥。持久製劑可以包括例如合適的聚合物或疏水性材料或離子交換樹脂。這種長效製劑對於預防性使用特別方便。The active compounds may also be formulated as a long-lasting formulation, either by intramuscular injection or by implantation, for example subcutaneous or intramuscular administration. Durable formulations may include, for example, suitable polymers or hydrophobic materials or ion exchange resins. Such long acting formulations are particularly convenient for prophylactic use.
呈現了適合於通過口腔進行肺部給藥的製劑，使得將含有活性化合物並期望具有0.5至7微米範圍內的直徑的顆粒在接受者的支氣管樹中遞送。Formulations suitable for pulmonary administration through the oral cavity are presented such that particles containing the active compound and desirably having a diameter in the range of 0.5 to 7 microns are delivered in the bronchial tree of the recipient.
作為一種可能性，這種製劑是以細粉碎粉末的形式，其可以方便地存在於適合於例如明膠的可刺穿的膠囊中，用於吸入裝置，或者可替換地作為包含活性化合物、合適的液體或氣體推進劑和任選的其它成分如表面活性劑和/或固體稀釋劑的自推進製劑。合適的液體推進劑包括丙烷和氯氟烴，且合適的氣體推進劑包括二氧化碳。還可以採用其中活性化合物以溶液或懸浮液的液滴的形式進行分配的自推進製劑。As a possibility, such a preparation is in the form of a finely pulverized powder which can conveniently be present in a pierceable capsule suitable for, for example, gelatin, for use in an inhalation device, or alternatively as an active compound, suitable A self-propelled formulation of a liquid or gaseous propellant and optionally other ingredients such as surfactants and/or solid diluents. Suitable liquid propellants include propane and chlorofluorocarbons, and suitable gas propellants include carbon dioxide. Self-propelled formulations in which the active compound is dispensed in the form of droplets of solution or suspension may also be employed.
這類自推進製劑類似於本領域已知的那些自推進製劑，並且可以通過已建立的方法製備。適當地，它們呈現在容器中，該容器提供有具有所需噴霧特性的手動操作或自動功能的閥；有利地，在每次操作該閥時，其具有遞送固定體積，例如25至100微升的計量式。Such self-propelled formulations are similar to those known in the art and can be prepared by established methods. Suitably, they are presented in a container provided with a manually operated or automatically functioning valve having the desired spray characteristics; advantageously, each time the valve is operated, it has a delivery fixed volume, for example 25 to 100 microliters Measured.
作為另外的可能性，活性化合物可以是用於在霧化器或噴霧器中使用的溶液或懸浮液的形式，由此採用加速氣流或超音波攪拌以產生用於吸入的細小液滴霧。As a further possibility, the active compound may be in the form of a solution or suspension for use in an atomizer or nebulizer, whereby accelerated gas flow or ultrasonic agitation is employed to create a fine droplet mist for inhalation.
適用於鼻內給藥的製劑包括通常類似於上述用於肺部給藥的製劑。當將製劑分配時，這類製劑應期望地具有在10至200微米範圍內的粒徑以使得能夠保持在鼻腔中；這可以通過適當地使用合適細微性的粉末或選擇適當的閥而實現。其它合適的製劑包括粒徑在20-500微米範圍內的粗粉末，用於通過從靠近鼻子的容器通過鼻通道而快速吸入來給藥，以及包含0.2至5%w/v的水性或油性溶液或懸浮液中的活性化合物的滴鼻液。Formulations suitable for intranasal administration include those generally similar to those described above for pulmonary administration. When formulating the formulation, such formulations should desirably have a particle size in the range of 10 to 200 microns to enable retention in the nasal cavity; this can be achieved by appropriate use of a suitable fine powder or by selection of a suitable valve. Other suitable formulations include coarse powders having a particle size in the range of 20-500 microns for administration by rapid inhalation through a nasal passage from a container near the nose, and containing 0.2 to 5% w/v of an aqueous or oily solution. Or nasal drops of the active compound in suspension.
藥學上可接受的載體是本領域技術人員熟知的，包括但不限於0.1 M且較佳0.05 M的磷酸鹽緩衝液或0.8%的飽和食鹽水。另外，這些藥學上可接受的載體可以是水性或非水性溶液、懸浮液和乳液。非水性溶劑的實例是丙二醇、聚乙二醇、植物油（如橄欖油）和可注射的有機酯（如油酸乙酯）。水性載體包括水、醇/水溶液、乳液或懸浮液，包括飽和食鹽水和緩衝媒介。腸胃外溶劑包括氯化鈉溶液、林格氏葡萄糖（Ringer's dextrose）、葡萄糖和氯化鈉、乳酸林格氏液（lactated Ringer's）或固定油。還可以存在防腐劑和其它添加劑，例如，如抗微生物劑、抗氧化劑、螯合劑、惰性氣體等。Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.1 M and preferably 0.05 M phosphate buffer or 0.8% saturated saline. Additionally, these pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saturated saline and buffering vehicles. Parenteral solvents include sodium chloride solution, Ringer's dextrose, glucose and sodium chloride, lactated Ringer's or fixed oils. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases, and the like.
可以提供適合於局部製劑的製劑，例如凝膠、乳膏劑或軟膏劑。這類製劑可以例如應用於傷口或潰瘍，將其直接地塗在傷口或潰瘍表面，或使其攜帶在可應用於待被治療區域上方的合適支撐物，如繃帶、紗布、網絲等上。Formulations suitable for topical formulations, such as gels, creams or ointments, may be provided. Such formulations may, for example, be applied to wounds or ulcers, applied directly to the wound or ulcer surface, or carried on a suitable support, such as a bandage, gauze, mesh, or the like, that can be applied over the area to be treated.
還可以提供液體或粉末製劑，可以將其直接噴灑或噴撒到待治療部位，例如，傷口或潰瘍上。可替換地，可以將如繃帶、紗布、網絲等的載體噴灑或噴撒有製劑，然後應用於待被治療的部位。Liquid or powder formulations may also be provided which may be sprayed or sprayed directly onto the site to be treated, for example, a wound or ulcer. Alternatively, a carrier such as a bandage, gauze, mesh or the like may be sprayed or sprayed with a preparation and then applied to the site to be treated.
根據本發明的另一方面，提供了用於製備如上所述的藥物或獸醫組合物的方法，該方法包括使活性化合物與載體聯合，例如通過混合。According to another aspect of the invention, there is provided a method for the preparation of a pharmaceutical or veterinary composition as described above, which method comprises combining an active compound with a carrier, for example by mixing.
通常，通過將活性劑與液體載體或細分散的固體載體或兩者均勻且緊密地聯合，然後如果需要的話，將產品成型來製備製劑。本發明涉及用於製備藥物組合物的方法，包括使式（I）的化合物與藥學上或獸醫學上可接受的載體或溶劑結合或聯合。鹽 / 酯 In general, the preparation is prepared by uniformly and intimately combining the active agent with a liquid carrier or a finely divided solid carrier or both, and then molding the product if necessary. The present invention relates to a process for the preparation of a pharmaceutical composition comprising combining or combining a compound of formula (I) with a pharmaceutically or veterinarily acceptable carrier or solvent. Salt / ester
本發明的化合物可以呈現為鹽或酯，特別是藥學上和獸醫學上可接受的鹽或酯。The compounds of the invention may be presented as salts or esters, especially pharmaceutically and veterinary acceptable salts or esters.
本發明化合物的藥學上可接受的鹽包括其合適的酸加成鹽或堿鹽。關於合適的藥物鹽的綜述可以在Berge等人，J Pharm Sci，66,199（1977）中找到。例如用強無機酸，如礦物酸，例如氫鹵酸（如鹽酸、氫溴酸和氫碘酸）、硫酸、磷酸硫酸鹽、硫酸氫鹽、半硫酸鹽、硫氰酸鹽、過硫酸鹽和磺酸；用強有機羧酸，如未取代或取代的（例如，通過鹵素）1至4個碳原子的鏈烷羧酸，如乙酸；用飽和或不飽和二羧酸，例如草酸、丙二酸、琥珀酸、馬來酸、富馬酸、鄰苯二甲酸或四鄰苯二甲酸；用羥基羧酸，例如抗壞血酸、乙醇酸、乳酸、蘋果酸、酒石酸或檸檬酸；用氨基酸，例如天冬氨酸或谷氨酸；用苯甲酸；或用有機磺酸，如未取代或取代的（例如，通過鹵素）(C1 -C4 )-烷基-或芳基-磺酸，如甲烷-或對甲苯磺酸，來形成鹽。不是藥學上或獸醫學上可接受的鹽可能仍然是有價值的中間體。Pharmaceutically acceptable salts of the compounds of the invention include suitable acid addition or phosphonium salts thereof. A review of suitable pharmaceutical salts can be found in Berge et al, J Pharm Sci, 66, 199 (1977). For example, strong inorganic acids such as mineral acids such as hydrohalic acids (such as hydrochloric acid, hydrobromic acid and hydroiodic acid), sulfuric acid, phosphoric acid sulfates, hydrogen sulfates, hemisulfates, thiocyanates, persulfates and a sulfonic acid; a strong organic carboxylic acid such as an unsubstituted or substituted (for example, by halogen) alkanecarboxylic acid having 1 to 4 carbon atoms, such as acetic acid; with a saturated or unsaturated dicarboxylic acid such as oxalic acid or propylene Acid, succinic acid, maleic acid, fumaric acid, phthalic acid or tetraphthalic acid; with hydroxycarboxylic acid, such as ascorbic acid, glycolic acid, lactic acid, malic acid, tartaric acid or citric acid; with amino acids, such as aspartic Acid or glutamic acid; with benzoic acid; or with an organic sulfonic acid, such as unsubstituted or substituted (for example, by halogen) (C 1 -C 4 )-alkyl- or aryl-sulfonic acid, such as methane - Or p-toluenesulfonic acid to form a salt. Salts that are not pharmaceutically or veterinary acceptable may still be valuable intermediates.
較佳的鹽包括，例如乙酸鹽、三氟乙酸鹽、乳酸鹽、葡萄糖酸鹽、檸檬酸鹽、酒石酸鹽、馬來酸鹽、蘋果酸鹽、泛酸鹽、己二酸鹽、藻酸鹽、天冬氨酸鹽、苯甲酸鹽、丁酸鹽、二葡糖酸鹽、環戊酸鹽、葡庚糖酸鹽、甘油磷酸鹽、草酸鹽、庚酸鹽、己酸鹽、富馬酸鹽、煙酸鹽、棕櫚酸酯、果膠酸鹽、3-苯基丙酸鹽、苦味酸鹽、新戊酸鹽、丙酸鹽、酒石酸鹽、乳糖酸鹽、pivolate、樟腦酸鹽、十一酸鹽和琥珀酸鹽，有機磺酸如甲磺酸鹽、乙磺酸鹽、2-羥基乙烷磺酸鹽、樟腦磺酸鹽、2-萘磺酸鹽、苯磺酸鹽、對氯苯磺酸鹽和對甲苯磺酸鹽；和無機酸如鹽酸、氫溴酸、氫碘酸、硫酸、硫酸氫、半硫酸、硫氰酸、過硫酸、磷酸和磺酸。Preferred salts include, for example, acetate, trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate, adipate, alginate , aspartate, benzoate, butyrate, digluconate, cyclopentanoate, glucoheptonate, glycerol phosphate, oxalate, heptanoate, hexanoate, rich Citrate, nicotinate, palmitate, pectate, 3-phenylpropionate, picrate, pivalate, propionate, tartrate, lactobionate, pivolate, camphorate , eleven acid and succinate, organic sulfonic acid such as methanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, camphorsulfonate, 2-naphthalenesulfonate, besylate, P-chlorobenzenesulfonate and p-toluenesulfonate; and inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, hydrogen sulfate, hemisulfuric acid, thiocyanic acid, persulfuric acid, phosphoric acid, and sulfonic acid.
根據被酯化的官能團，使用有機酸或醇/氫氧化物形成酯。有機酸包括羧酸，如未取代或取代的（例如，通過鹵素）1至12個碳原子的鏈烷羧酸，如乙酸；用飽和或不飽和二羧酸，例如草酸、丙二酸、琥珀酸、馬來酸、富馬酸、鄰苯二甲酸或四鄰苯二甲酸；用羥基羧酸，例如抗壞血酸、乙醇酸、乳酸、蘋果酸、酒石酸或檸檬酸；用氨基酸，例如天冬氨酸或谷氨酸；用苯甲酸；或用有機磺酸，如未取代或取代的（例如，通過鹵素）(C1 -C4 )-烷基-或芳基-磺酸，如甲烷-或對甲苯磺酸。合適的氫氧化物包括無機氫氧化物，如氫氧化鈉、氫氧化鉀、氫氧化鈣、氫氧化鋁。醇包括可以是未取代或取代的（例如，通過鹵素）1-12個碳原子的烷醇。對映異構體 / 互變異構體 An ester is formed using an organic acid or an alcohol/hydroxide depending on the functional group to be esterified. The organic acid includes a carboxylic acid such as an unsubstituted or substituted (for example, by halogen) alkanecarboxylic acid having 1 to 12 carbon atoms, such as acetic acid; with a saturated or unsaturated dicarboxylic acid such as oxalic acid, malonic acid, amber. Acid, maleic acid, fumaric acid, phthalic acid or tetraphthalic acid; with hydroxycarboxylic acids such as ascorbic acid, glycolic acid, lactic acid, malic acid, tartaric acid or citric acid; with amino acids such as aspartic acid or Glutamate; with benzoic acid; or with an organic sulfonic acid, such as unsubstituted or substituted (for example, by halogen) (C 1 -C 4 )-alkyl- or aryl-sulfonic acid, such as methane- or p-toluene Sulfonic acid. Suitable hydroxides include inorganic hydroxides such as sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide. Alcohols include alkanols which may be unsubstituted or substituted (e.g., by halogen) from 1 to 12 carbon atoms. Enantiomer / tautomer
在先前討論的本發明的所有方面中，在適當的情況下，本發明包括本發明化合物的所有對映異構體、非對映異構體和互變異構體。本領域技術人員應識別具有光學性質（一個以上的手性碳原子）或互變異構特徵的化合物。可以通過本領域已知的方法分離/製備相應的對映異構體和/或互變異構體。In all aspects of the invention previously discussed, the invention includes all enantiomers, diastereomers and tautomers of the compounds of the invention, where appropriate. Those skilled in the art will recognize compounds having optical properties (more than one chiral carbon atom) or tautomeric characteristics. The corresponding enantiomers and/or tautomers can be separated/prepared by methods known in the art.
對映異構體以其手性中心的絕對構型為特徵，並由Cahn、Ingold和Prelog的R -和S -排序規則進行描述。這些慣例在本領域中是熟知的（例如，參見“Advanced Organic Chemistry”，第3版，March，J.，John Wiley和Sons，New York，1985）。Enantiomers are characterized by the absolute configuration of their chiral centers and are described by the R- and S -sorting rules of Cahn, Ingold and Prelog. These conventions are well known in the art (for example, see "Advanced Organic Chemistry", 3rd edition, March, J., John Wiley and Sons, New York, 1985).
含有手性中心的本發明化合物可以用作為外消旋混合物、富含對映異構體的混合物，或者可以使用熟知的技術分離外消旋混合物，並且可以單獨地使用單個對映異構體。立體異構體和幾何異構體 The compounds of the invention containing a chiral center can be used as a racemic mixture, a mixture enriched in enantiomers, or the racemic mixture can be separated using well known techniques, and the individual enantiomers can be used separately. Stereoisomers and geometric isomers
一些本發明的化合物可以作為立體異構體和/或幾何異構體存在 - 例如，它們可以具有一個以上的不對稱和/或幾何中心，因此可以以兩種以上的立體異構和/或幾何形式存在。本發明考慮了使用所有的這些抑制劑的單個立體異構體和幾何異構體及其混合物。權利要求中使用的術語包括這些形式，隻要該形式保持適當的功能活性（儘管不一定以相同程度）。Some of the compounds of the invention may exist as stereoisomers and/or geometric isomers - for example, they may have more than one asymmetric and/or geometric center, and thus may be more than two stereoisomers and/or geometries Form exists. The present invention contemplates the use of all stereoisomers and geometric isomers of these inhibitors, and mixtures thereof. The terms used in the claims include these forms as long as the form retains the appropriate functional activity (although not necessarily to the same extent).
本發明還包括藥劑或其藥學上可接受的鹽的所有合適的同位素變體。將本發明的藥劑或其藥學上可接受的鹽的同位素變體定義為其中至少一個原子被具有相同原子數但原子品質與通常在自然界中發現的原子的原子品質不同的原子取代的一種。可以併入該藥劑及其藥學上可接受的鹽的同位素的實例包括氫、碳、氮、氧、磷、硫、氟和氯的同位素，如分別地2 H、3 H、13 C、14 C、15 N、17 O、18 O、31 P、32 P、35 S、18 F和36 Cl。藥劑和藥學上可接受的鹽的某些同位素變體，例如其中併入如3 H或14 C的放射性同位素的那些同位素變體，可用於藥物和/或底物組織分佈研究。氚標記的，即3 H，和碳-14，即14 C同位素，由於其易於製備和可檢測性是特別佳的。此外，用同位素如氘（即2 H）取代可以提供由更大的代謝穩定性，例如增加的體內半衰期或降低的劑量需求產生的某些治療優勢，因此在某些情況下可能是較佳的。例如，本發明包括其中任一氫原子被氘原子代替的通式（I）的化合物。通常可以使用合適試劑的適當同位素變體由常規方法製備本發明的藥劑和本發明的其藥學上可接受的鹽的同位素變體。前藥 The invention also includes all suitable isotopic variations of the agent or a pharmaceutically acceptable salt thereof. An isotopic variation of an agent of the present invention or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is substituted with an atom having the same atomic number but an atomic quality different from that of an atom generally found in nature. Examples of isotopes which may be incorporated into the agent and its pharmaceutically acceptable salts include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as 2 H, 3 H, 13 C, 14 C, respectively. , 15 N, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F and 36 Cl. Certain isotopic variations of agents and pharmaceutically acceptable salts, such as those in which a radioisotope such as 3 H or 14 C is incorporated, are useful in drug and/or substrate tissue distribution studies. The ruthenium-labeled, ie, 3 H, and carbon-14, 14 C isotope, are particularly preferred due to their ease of preparation and detectability. In addition, substitution with isotopes such as deuterium (ie, 2 H) may provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and thus may be preferred in some circumstances. . For example, the invention includes compounds of formula (I) wherein any of the hydrogen atoms are replaced by a deuterium atom. Isotopic variations of the agents of the invention and the pharmaceutically acceptable salts thereof of the invention can generally be prepared by conventional methods using appropriate isotopic variations of the appropriate reagents. Prodrug
本發明還包括以前藥形式的本發明化合物，即在體內釋放根據通式（I）的活性母體藥物的共價鍵合的化合物。這樣的前藥通常是其中一個以上的適當基團已被修飾，使得在給藥至人或哺乳動物受試者後該修飾可能被逆轉的本發明化合物。通常通過這類受試者中天然存在的酶來進行該逆轉，儘管可能將第二藥劑與這種前藥一起給藥以便在體內進行反轉。這類修飾的實例包括酯（例如，上述的任何一種），其中可以由酯酶等進行這種逆轉。其它的這類系統對於本領域技術人員來說是熟知的。溶劑化物 The invention also includes a compound of the invention in a prodrug form, i.e., a compound that releases a covalent linkage of an active parent drug according to formula (I) in vivo. Such prodrugs are generally compounds of the invention in which one or more suitable groups have been modified such that the modification may be reversed after administration to a human or mammalian subject. This reversal is typically performed by an enzyme naturally present in such subjects, although it is possible to administer a second agent with such prodrug for inversion in vivo. Examples of such modifications include esters (for example, any of the above) in which such reversal can be carried out by esterase or the like. Other such systems are well known to those skilled in the art. Solvate
本發明還包括本發明化合物的溶劑化物形式。權利要求中使用的術語包括這些形式。多晶型 The invention also includes solvated forms of the compounds of the invention. The terms used in the claims include these forms. Polymorph
本發明還涉及以本發明化合物的各種結晶形式、多晶形式和水合形式的本發明化合物。在製藥工業中已經確定了，可以通過稍微改變在這類化合物的合成製備中使用的溶劑的純化和/或分離的方法而以任何這些形式分離化學化合物。給藥 The invention further relates to compounds of the invention in various crystalline, polymorphic and hydrated forms of the compounds of the invention. It has been determined in the pharmaceutical industry that chemical compounds can be isolated in any of these forms by slightly altering the purification and/or separation of the solvents used in the synthetic preparation of such compounds. Administration
本發明的藥物組合物可適用於直腸、鼻內、支氣管內、局部（包括口腔和舌下）、***或腸胃外（包括皮下、肌內、靜脈內、動脈內和皮內）、腹膜內或鞘內給藥。較佳地，製劑是口服給藥的製劑。製劑可以方便地以單位劑型，即以包含單位劑量或者單位劑量的多個單位或子單位的離散部分的形式呈現。作為實例，製劑可以是以片劑和緩釋膠囊的形式，並且可以通過藥學領域熟知的任何方法進行製備。The pharmaceutical composition of the present invention can be applied to the rectum, intranasal, intrabronchial, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intraarterial and intradermal), intraperitoneal or Intrathecal administration. Preferably, the formulation is a formulation for oral administration. Formulations may conveniently be presented in unit dosage form, i.e., in discrete portions comprising a plurality of units or subunits in unit dosages or unit dosages. As an example, the formulation may be in the form of a tablet and a sustained release capsule, and may be prepared by any method well known in the art of pharmacy.
本發明的用於口服給藥的製劑可以呈現為：離散單位，如各自含有預定量的活性劑的膠囊、凝膠劑、滴劑、扁囊劑、丸劑或片劑；作為粉末或顆粒；作為水性液體或非水性液體中的活性劑的溶液、乳液或懸浮液；或作為水包油液體乳劑或油包水液體乳劑；或作為彈丸注射等。較佳地，每劑量的這些組合物含有1至250 mg且更佳10-100 mg的活性成分。The preparation for oral administration of the present invention may be presented as discrete units such as capsules, gels, drops, cachets, pills or tablets each containing a predetermined amount of the active agent; as a powder or granule; a solution, emulsion or suspension of the active agent in an aqueous liquid or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; or as a bolus injection or the like. Preferably, each of these compositions contains from 1 to 250 mg and more preferably from 10 to 100 mg of active ingredient per dose.
對於用於口服給藥的組合物（例如，片劑和膠囊），術語“可接受的載體”包括溶劑，如常用輔料，例如黏合劑，例如糖漿、***膠、明膠、山梨糖醇、黃芪膠、聚乙烯吡咯烷酮（聚維酮）、甲基纖維素、乙基纖維素、羧甲基纖維素鈉、羥丙基甲基纖維素、蔗糖和澱粉；填料和載體，例如玉米澱粉、明膠、乳糖、蔗糖、微晶纖維素、高嶺土、甘露醇、磷酸二鈣、氯化鈉和海藻酸；和潤滑劑，如硬脂酸鎂、硬脂酸鈉和其它金屬硬脂酸酯、甘油硬脂酸、硬脂酸、矽酮流體、滑石蠟、油和膠體二氧化矽。調味劑如薄荷、冬青油、櫻桃香料等也可以使用。可能需要添加著色劑以使劑型容易被識別。片劑也可以通過本領域熟知的方法進行包衣。For compositions for oral administration (eg, tablets and capsules), the term "acceptable carrier" includes solvents such as conventional excipients such as binders such as syrup, acacia, gelatin, sorbitol, tragacanth. , polyvinylpyrrolidone (povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, such as corn starch, gelatin, lactose , sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metal stearates, glyceryl stearic acid , stearic acid, anthrone fluid, talc, oil and colloidal cerium oxide. Flavoring agents such as peppermint, wintergreen oil, cherry flavor, and the like can also be used. Colorants may need to be added to make the dosage form readily identifiable. Tablets can also be coated by methods well known in the art.
可以通過壓製或模製，任選地與一種以上的輔助成分一起製成片劑。可以通過在合適的機器中壓製處於自由流動形式（如粉末或顆粒）的活性劑，任選地與黏合劑、潤滑劑、惰性稀釋劑、潤滑物質、防腐劑、表面活性劑或分散劑混合來製備壓製片劑。可以通過在合適的機器中模製用惰性液體稀釋劑潤濕的粉末化合物的混合物來製備模製片劑。可以將片劑任選地包衣或刻痕，並且可以進行配製從而提供活性劑的緩慢的或受控的釋放。Tablets can be made by compression or molding, optionally with more than one accessory ingredient. The active agent in a free-flowing form such as a powder or granules may optionally be compressed in a suitable machine, optionally mixed with a binder, lubricant, inert diluent, lubricating substance, preservative, surfactant or dispersing agent. Compressed tablets were prepared. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated to provide a slow or controlled release of the active agent.
適用於口服給藥的其它製劑包括包含調味基質，通常為蔗糖和***膠或黃芪膠中的活性劑的錠劑；包含惰性基質如明膠和甘油，或蔗糖和***膠中的活性劑的軟錠劑；和包含合適液體載體中的活性劑的漱口水。Other formulations suitable for oral administration include lozenges comprising a flavoring base, typically an active agent in sucrose and gum arabic or tragacanth; soft lozenges comprising an inert matrix such as gelatin and glycerin, or active agents in sucrose and gum arabic And a mouthwash comprising an active agent in a suitable liquid carrier.
其它給藥形式包括可以進行靜脈內、動脈內、鞘內、皮下、皮內、腹膜內或肌肉內注射並由無菌或可滅菌溶液製備的溶液或乳劑。可注射形式通常每劑含有10-1000 mg，較佳10-250 mg之間的活性成分。Other forms of administration include solutions or emulsions which can be administered intravenously, intraarterially, intrathecally, subcutaneously, intradermally, intraperitoneally or intramuscularly and prepared from sterile or sterilizable solutions. The injectable form will usually contain from 10 to 1000 mg, preferably from 10 to 250 mg, of active ingredient per dose.
本發明的藥物組合物還可以是以肛門塞藥、***栓劑、懸浮液、乳劑、洗劑、軟膏劑、乳膏劑、凝膠劑、噴霧劑、溶液或撒布劑的形式。The pharmaceutical compositions of the invention may also be in the form of an anal plug, pessary, suspension, emulsion, lotion, ointment, cream, gel, spray, solution or spread.
透皮給藥的另一種方法是使用皮膚貼劑。例如，可以將活性成分摻入由聚乙二醇或液體石蠟的水性乳液組成的乳膏劑中。活性成分還可以以1至10重量%的濃度摻入由白蠟或白色軟石蠟基質以及可能需要的穩定劑和防腐劑組成的軟膏劑中。劑量 Another method of transdermal administration is the use of a skin patch. For example, the active ingredient can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycol or liquid paraffin. The active ingredient can also be incorporated in an ointment consisting of a white wax or a white soft paraffin base and possibly a stabilizer and preservative at a concentration of from 1 to 10% by weight. dose
本領域普通技術人員可以容易地確定本發明組合物之一的給予至受試者的合適劑量，而無需過多的實驗。通常，醫師將確定最適合個體患者的實際劑量，並且該劑量將取決於多種因素，包括所用特定化合物的活性、該化合物的代謝穩定性和作用時間、年齡、體重、常規健康狀況、性別、飲食、給藥方式和時間、***速率、藥物組合、特定病情的嚴重程度以及接受治療的個體。本文揭露的劑量是平均病況的示例。當然，可以有個別情況，這種情況中的更高或更低劑量範圍是有價值的，並且這種劑量範圍在本發明的範圍內。One of ordinary skill in the art can readily determine the appropriate dosage of one of the compositions of the present invention to the subject without undue experimentation. Generally, the physician will determine the actual dosage that is most suitable for the individual patient, and the dosage will depend on a variety of factors, including the activity of the particular compound employed, the metabolic stability and duration of action of the compound, age, weight, general health, sex, diet. , mode of administration and time, rate of excretion, combination of drugs, severity of a particular condition, and the individual receiving treatment. The dosages disclosed herein are examples of average conditions. Of course, there may be individual cases where higher or lower dosage ranges are valuable and such dosage ranges are within the scope of the invention.
根據本發明，可以給予有效量的通式（I）化合物以抑制與特定病情或疾病有關的激酶。當然，根據化合物的給藥類型，將進一步改變劑量。例如，較佳將通式（I）的化合物腸胃外給藥，以達到急性治療的“有效量”。儘管肌內彈丸注射也是有用的，但是靜脈輸注5%葡萄糖水溶液或生理飽和食鹽水中的化合物或具有合適輔料的類似製劑是最有效的。通常，腸胃外劑量為約0.01至約100 mg/kg；較佳在0.1至20 mg/kg之間，以這種方式維持血漿中藥物濃度在有效抑制激酶的濃度。可以以一水準每天給予化合物一至四次，達到約0.4至約400 mg/kg/天的總日劑量。本領域普通技術人員通過將藥劑的血液水準與具有治療效果的所需濃度相比，容易確定治療有效的本發明化合物的精確量，以及將該化合物最佳給藥的途徑。In accordance with the present invention, an effective amount of a compound of formula (I) can be administered to inhibit a kinase associated with a particular condition or disease. Of course, the dosage will be further varied depending on the type of administration of the compound. For example, a compound of formula (I) is preferably administered parenterally to achieve an "effective amount" of acute treatment. Although intramuscular bolus injection is also useful, intravenous infusion of a compound in 5% dextrose or physiologically saturated saline or a similar formulation with suitable excipients is most effective. Typically, the parenteral dosage is from about 0.01 to about 100 mg/kg; preferably between 0.1 and 20 mg/kg, in such a manner that the concentration of the drug in the plasma is maintained to effectively inhibit the concentration of the kinase. The compound can be administered from one to four times a day to a total daily dose of from about 0.4 to about 400 mg/kg/day. One of ordinary skill in the art can readily determine the precise amount of a therapeutically effective compound of the invention, as well as the route of optimal administration of the compound, by comparing the blood level of the agent to the desired concentration of the therapeutic effect.
也可以向患者口服給予本發明化合物，以藥物濃度能夠足以實現治療一種以上的本文揭露的治療適應症的方式。通常，以與患者病情一致的方式，以約0.1至約50 mg/kg之間的口服劑量給予含有該化合物的藥物組合物。口服劑量較佳為約0.5至約20 mg/kg。The compounds of the invention may also be administered orally to a patient at a drug concentration sufficient to effect treatment of more than one of the therapeutic indications disclosed herein. Generally, a pharmaceutical composition containing the compound is administered in an oral dose of between about 0.1 and about 50 mg/kg in a manner consistent with the condition of the patient. The oral dose is preferably from about 0.5 to about 20 mg/kg.
當根據本發明給予本發明化合物時，預期不會產生不可接受的毒理作用。可以以多種生物測定法之一測試可以具有良好生物利用度的本發明化合物，以確定具有給定藥理作用所需的化合物的濃度。組合 When a compound of the invention is administered in accordance with the invention, no unacceptable toxicological effects are expected. Compounds of the invention which may have good bioavailability may be tested in one of a variety of bioassays to determine the concentration of a compound required for a given pharmacological effect. combination
在一個特別佳的實施方式中，將一種以上的本發明化合物與一種以上的其它活性劑（例如，市場上可獲得的現有藥物）組合給藥。在這種情況下，可以將本發化合物與一種以上的其它活性劑連續、同時或序貫給藥。In a particularly preferred embodiment, more than one compound of the invention is administered in combination with more than one other active agent (e.g., a commercially available drug). In this case, the present compound can be administered continuously, simultaneously or sequentially with more than one other active agent.
通常，藥物在組合使用時更有效。特別地，組合治療是期望的，以便避免主要毒性、作用機制和抗藥性機制的重疊。此外，還期望以最大耐受劑量和這種劑量之間的最小時間間隔給予大多數藥物。組合化療藥物的主要優點是其可以通過生物化學相互作用促進附加的或可能的協同作用，並且還可能減少出現抗藥性。Generally, drugs are more effective when used in combination. In particular, combination therapies are desirable in order to avoid overlap of major toxicity, mechanism of action and mechanism of resistance. In addition, it is also desirable to administer most drugs at a maximum tolerated dose and a minimum time interval between such doses. The main advantage of combination chemotherapy drugs is that they can promote additional or possible synergistic effects through biochemical interactions, and may also reduce the emergence of drug resistance.
可以通過研究測試化合物與已知或猜測為在治療特定病症中有價值的藥劑的抑制活性來提出有益的組合。該方法也可用於確定這種藥劑的給藥順序，即在遞送化合物之前、與之同時或之後。這種給藥方式可以是本文所鑒定的所有活性劑的特徵。試驗 A beneficial combination can be proposed by studying the inhibitory activity of a test compound with an agent known or suspected to be valuable in treating a particular condition. The method can also be used to determine the order of administration of such agents, ie, before, simultaneously with, or after delivery of the compound. This mode of administration can be characteristic of all active agents identified herein. test
本發明的另一方面涉及如上所述的化合物在試驗中的用途，這種試驗用於鑒定能夠抑制一種以上的激酶、更佳LRRK、甚至更佳LRRK2的其它候選化合物。Another aspect of the invention relates to the use of a compound as described above in an assay for identifying other candidate compounds capable of inhibiting more than one kinase, more preferably LRRK, even better LRRK2.
較佳地，該試驗是競爭性結合試驗。Preferably, the test is a competitive binding assay.
更佳地，競爭性結合試驗包括讓本發明化合物與激酶、較佳LRRK、更佳LRRK2和候選化合物接觸，並檢測根據本發明的化合物與激酶之間的相互作用的任何變化。More preferably, the competitive binding assay involves contacting a compound of the invention with a kinase, preferably LRRK, a better LRRK2 and a candidate compound, and detecting any change in the interaction between the compound according to the invention and the kinase.
較佳地，通過本發明化合物的常規SAR修飾產生候選化合物。Preferably, the candidate compound is produced by conventional SAR modification of the compounds of the invention.
如本文所使用的，術語“常規SAR修飾”是指通過化學衍生化來改變給定化合物的本領域已知的標準方法。As used herein, the term "conventional SAR modification" refers to standard methods known in the art for modifying a given compound by chemical derivatization.
因此，在一個方面，所鑒定的化合物可以作為模型（例如，範本），用於開發其它化合物。在這種測試中使用的化合物可以游離於溶液中、固定在固體載體上、承載在細胞表面或位於細胞內。可以測量化合物和待測試藥劑之間的活性消除或結合複合物的形成。Thus, in one aspect, the identified compounds can be used as models (eg, templates) for the development of other compounds. The compounds used in this test can be freed in solution, immobilized on a solid support, carried on the cell surface or located within the cell. The elimination of activity or the formation of a binding complex between the compound and the agent to be tested can be measured.
本發明的試驗可以是篩選，因而測試了大量藥劑。在一方面，本發明的測定方法是高通量篩選。The assay of the present invention can be screening, thus testing a large number of agents. In one aspect, the assay of the invention is a high throughput screen.
本發明還考慮了使用競爭性藥物篩選試驗，其中能夠結合化合物的中和抗體與用於結合化合物的測試化合物特異性競爭。The present invention also contemplates the use of competitive drug screening assays in which a neutralizing antibody capable of binding a compound specifically competes with a test compound for binding the compound.
提供了用於篩選的另一技術，用於對物質具有合適結合親和力的試劑進行高通量篩選（HTS），並且該技術基於WO 84/03564中詳細描述的方法。Another technique for screening is provided for high throughput screening (HTS) of reagents having suitable binding affinities for the substance, and the technique is based on the method detailed in WO 84/03564.
預期本發明的測定方法，將適合於對測試化合物進行小規模篩選和大規模篩選以及適合於定量試驗。It is expected that the assay method of the present invention will be suitable for small scale screening and large scale screening of test compounds as well as for quantitative assays.
較佳地，競爭性結合試驗包括在激酶的已知底物存在下，讓本發明化合物與該激酶接觸，並檢測該激酶和該已知底物之間相互作用的任何變化。Preferably, competitive binding assays involve contacting a compound of the invention with the kinase in the presence of a known substrate for the kinase and detecting any change in the interaction between the kinase and the known substrate.
本發明的另一方面提供了檢測配體與激酶結合的方法，該方法包括以下步驟： （i）在激酶的已知底物存在下，讓配體與該激酶接觸； （ii）檢測該激酶和該已知底物之間的相互作用的任何變化； 並且其中該配體是本發明化合物。Another aspect of the invention provides a method of detecting binding of a ligand to a kinase, the method comprising the steps of: (i) contacting a ligand with the kinase in the presence of a known substrate of a kinase; (ii) detecting the kinase Any change in interaction with the known substrate; and wherein the ligand is a compound of the invention.
本發明的一個方面涉及一種方法，包括以下步驟： （a）進行上述測定方法； （b）鑒定能夠與配體結合結構域結合的一種以上的配體；和 （c）製備一定量的該一種以上的配體。One aspect of the invention relates to a method comprising the steps of: (a) performing the above assay; (b) identifying one or more ligands capable of binding to a ligand binding domain; and (c) preparing a quantity of the one The above ligands.
本發明的另一方面提供了一種方法，包括以下步驟： （a）進行上述測定方法； （b）鑒定能夠與配體結合結構域結合的一種以上的配體；和 （c）製備包含該一種以上的配體的藥物組合物。Another aspect of the invention provides a method comprising the steps of: (a) performing the above assay; (b) identifying one or more ligands capable of binding to a ligand binding domain; and (c) preparing comprising the one A pharmaceutical composition of the above ligand.
本發明的另一方面提供了一種方法，包括以下步驟： （a）進行上述測定方法； （b）鑒定能夠與配體結合結構域結合的一種以上的配體； （c）修飾能夠與配體結合結構域結合的該一種以上的配體； （d）進行這種上述測定方法； （e）任選地製備包含該一種以上的配體的藥物組合物。Another aspect of the invention provides a method comprising the steps of: (a) performing the above assay; (b) identifying one or more ligands capable of binding to a ligand binding domain; (c) modifying the ligand and the ligand The one or more ligands bound to the binding domain; (d) performing the above assay; (e) optionally preparing a pharmaceutical composition comprising the one or more ligands.
本發明還涉及通過上述方法鑒定的配體。The invention also relates to ligands identified by the above methods.
本發明的另一方面涉及包含通過上述方法鑒定的配體的藥物組合物。Another aspect of the invention relates to a pharmaceutical composition comprising a ligand identified by the above method.
本發明的另一方面涉及通過上述方法鑒定的配體在製備藥物組合物中的用途，該藥物組合物用於治療一種以上的病症。Another aspect of the invention relates to the use of a ligand identified by the above method for the preparation of a pharmaceutical composition for the treatment of more than one condition.
上述方法可用於篩選可用作一種以上的激酶的抑制劑的配體。The above methods can be used to screen ligands that can be used as inhibitors of more than one kinase.
通式（I）的化合物既可有用地作為實驗室工具又可有用地作為治療劑。在實驗室中，本發明的某些化合物可用於建立通常被稱為“靶標驗證”的方法，在疾病狀態的確定或進展期間，已知或新發現的激酶是否導致關鍵性的或至少重要的生物化學功能產生。常規的合成方法 The compounds of formula (I) are useful both as laboratory tools and useful as therapeutic agents. In the laboratory, certain compounds of the invention can be used to establish a method commonly referred to as "target validation," whether known or newly discovered kinases are critical or at least important during the determination or progression of a disease state. Biochemical function is produced. Conventional synthesis method
通過以下實施例描述本發明。但是應當理解，本發明不限於這些實施方式，這些實施例僅用於提出實施本發明的方法。The invention is described by the following examples. However, it should be understood that the invention is not limited to the embodiments, which are merely used to present a method of practicing the invention.
在整個說明書中使用以下縮寫： AcOH 乙酸 AlCl3 氯化鋁 BH3 硼烷 Bn 苄基 BuOH 正丁醇 CuI 碘化亞銅 DCM 二氯甲烷 DMF N,N-二甲基甲醯胺 DMSO 二甲基亞碸 DIEA, DIPEA N,N-二異丙基乙胺 EA 乙酸乙酯 EDCI 1-(3-二甲氨基丙基)-3-乙基碳二亞胺鹽酸鹽 EtOH 乙醇 EtOAc 乙酸乙酯 Et3 N 三乙胺 HATU 2-(7-偶氮苯並三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯 HOBT 羥基苯並*** I2 碘 IPA 異丙醇 KOAc 醋酸鉀 KOH 氫氧化鉀 K3 PO4 磷酸鉀 LiAlH4 氫化鋁鋰 LiCl 氯化鋰 LCMS 高效液相色譜-質譜聯用 MeOH 甲醇 MeCN 乙腈 MeI 碘甲烷 MsCl 甲磺醯氯 Na2 CO3 碳酸鈉 NaHCO3 碳酸氫鈉 Na2 S2 O3 硫代硫酸鈉 NaOH 氫氧化鈉 NaBH4 硼氫化鈉 (n-Bu)4 NI 四丁基碘化銨 n-BuLi 正丁基鋰 NH3 氨 NH4 Cl 氯化銨 NIS N-碘代琥珀醯亞胺 NMR 核磁共振 prep-HPLC 製備高效液相色譜 prep-TLC 製備薄層色譜 PMB 對甲氧基苄基 PMBCl 對甲氧基苄基氯 PPh3 三苯基膦 Pd(dppf)Cl2 1,1'-雙(二苯基膦基)二茂鐵]二氯化鈀（II） Pd(PPh3 )4 四（三苯基膦）鈀（0） Pd(OAc)2 乙酸鈀（II） PE 石油醚 rt 室溫 Sphos 2-二環己基膦基-2',6'-二甲氧基聯苯 t-BuOK 叔丁醇鉀t -BuONa 叔丁醇鈉 TEA 三乙胺 THF 四氫呋喃 TFA 三氟乙酸 Trt 三苯甲基 UV 紫外線The following abbreviations are used throughout the specification: AcOH Acetic acid AlCl 3 Aluminum chloride BH 3 Borane Bn Benzyl BuOH n-butanol CuI Cuprous iodide DCM Dichloromethane DMF N,N-dimethylformamide DMSO Dimethyl Acetone DIEA, DIPEA N, N-diisopropylethylamine EA Ethyl acetate EDCI 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EtOH Ethanol EtOAc Ethyl acetate Et 3 N triethylamine HATU 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluron hexafluorophosphate HOBT hydroxybenzotriazole I 2 iodine IPA isopropyl Alcohol KOAc potassium acetate KOH potassium hydroxide K 3 PO 4 potassium phosphate LiAlH 4 lithium aluminum hydride LiCl lithium chloride LCMS high performance liquid chromatography-mass spectrometry MeOH methanol MeCN acetonitrile MeI methyl iodide MsCl methanesulfonium chloride Na 2 CO 3 sodium carbonate NaHCO 3 sodium bicarbonate Na 2 S 2 O 3 sodium thiosulfate NaOH sodium hydroxide NaBH 4 sodium borohydride (n-Bu) 4 NI tetrabutylammonium iodide n-BuLi n-butyl lithium NH 3 ammonia NH 4 Cl Ammonium chloride NIS N-iodosuccinimide NMR nuclear magnetic resonance prep-HPLC preparative high performance liquid chromatography prep-TLC preparation thin layer chromatography PMB p-methoxybenzyl PMBCl p-methoxybenzyl chloride PPh 3 triphenyl phosphine Pd (dppf) Cl 2 1,1'- bis (diphenylphosphino) ferrocene ] Dichloropalladium (II) Pd (PPh 3) 4 tetrakis (triphenylphosphine) palladium (0) Pd (OAc) 2 palladium (II) acetate PE petroleum ether rt room temperature Sphos 2- dicyclohexylphosphino group - 2',6'-dimethoxybiphenyl t-BuOK potassium t -butoxide t- BuONa sodium tert-butoxide TEA triethylamine THF tetrahydrofuran TFA trifluoroacetic acid Trt trityl UV UV
方案 1 ： 
步驟1描述了將式A轉化為式B，其中X是鹵素，較佳溴或碘，並且LG是離去基團，如琥珀醯亞胺。Step 1 describes the conversion of Formula A to Formula B wherein X is a halogen, preferably bromine or iodine, and LG is a leaving group such as amber imine.
反應是在合適的溶劑中在合適的鹵化劑，如碘或N-溴代琥珀醯亞胺的存在下進行的，任選地在堿，如氫氧化鉀的存在下進行的。The reaction is carried out in a suitable solvent in the presence of a suitable halogenating agent, such as iodine or N-bromosuccinimide, optionally in the presence of hydrazine, such as potassium hydroxide.
典型條件（X=I），1,4-二氧六環中1當量的式A、2當量的I2 、3.7當量的KOH，在75℃下持續4小時。步驟 2 Typical conditions (X = I), 1 equivalent of Formula A, 2 equivalents of I 2 , 3.7 equivalents of KOH in 1,4-dioxane, were continued at 75 ° C for 4 hours. Step 2
步驟2描述了將式B轉化成式C，其中PG定義為保護基團，包括但不限於叔丁氧基羰基-；苄氧基羰基-；苄基-；4-甲氧基苄基-；2,4-二甲氧基苄基-或三苯甲基-；LG定義為離去基團，如鹵素或碳酸叔丁酯。Step 2 describes the conversion of formula B to formula C wherein PG is defined as a protecting group including, but not limited to, tert-butoxycarbonyl-; benzyloxycarbonyl-; benzyl-; 4-methoxybenzyl-; 2,4-Dimethoxybenzyl- or trityl-; LG is defined as a leaving group such as halogen or tert-butyl carbonate.
該反應包括用保護基團封端吡唑NH。本領域技術人員將會理解，許多保護基團可用於此目的（參見Greene，Theodora W.和Wuts，Peter G.M. Greene’s Protective Groups in Organic Synthesis. 第4版（2006））。技術人員還將理解，可以在N1或N2處引入保護基團，並且該比例可以根據PG的性質或展開的精確反應條件而改變。反應條件取決於保護基團的性質。The reaction involves capping the pyrazole NH with a protecting group. Those skilled in the art will appreciate that a number of protecting groups can be used for this purpose (see Greene, Theodora W. and Wuts, Peter G. M. Greene's Protective Groups in Organic Synthesis. 4th Ed. (2006)). The skilled person will also understand that a protecting group can be introduced at N1 or N2 and that the ratio can vary depending on the nature of the PG or the precise reaction conditions of the unfolding. The reaction conditions depend on the nature of the protecting group.
典型條件（PG=4-甲氧基苄基）：在室溫下將DMF中的1當量的4-甲氧基苄基氯； 1當量的式B，2當量的氫氧化鉀攪拌過夜。步驟 3 Typical conditions (PG = 4-methoxybenzyl): 1 equivalent of 4-methoxybenzyl chloride in DMF; 1 equivalent of Formula B, 2 equivalents of potassium hydroxide were stirred overnight at room temperature. Step 3
步驟3描述了將式C轉化成式D，其中X是鹵素，基團R1 可以任選地含有官能團，可以使用本領域技術人員已知的標準條件在合成方法的後續階段操作該官能團。Step 3 describes the conversion of Formula C to Formula D wherein X is a halogen, the group R 1 may optionally contain a functional group, which may be manipulated at a subsequent stage of the synthesis process using standard conditions known to those skilled in the art.
該反應包括讓式C中的氯基與合適溶劑中的氨基基團進行親核取代，任選地在質子酸（Bronsted acid）的存在下。該反應通常需要加熱，用熱的方法或使用微波照射進行加熱。This reaction involves nucleophilic substitution of the chloro group of formula C with an amino group in a suitable solvent, optionally in the presence of a protonic acid. The reaction usually requires heating and heating by thermal means or by microwave irradiation.
典型條件：將正丁醇中的2.5當量的胺、1當量的式C在密封的反應器中加熱至180℃持續5小時。步驟 4 Typical conditions: 2.5 equivalents of amine in n-butanol, 1 equivalent of Formula C were heated to 180 °C in a sealed reactor for 5 hours. Step 4
步驟4涉及將式D轉化為式E。X是鹵素，且較佳碘。該反應涉及在合適的過渡金屬催化劑和合適的堿（較佳三乙胺）以及任選的額外添加劑（如四丁基碘化銨）的存在下，取代的乙烯基酯與式D的交叉偶聯。本領域技術人員通常將這種類型的轉化稱為“Heck反應”。Step 4 involves converting Formula D to Formula E. X is a halogen and is preferably iodine. The reaction involves the cross-over of the substituted vinyl ester with Formula D in the presence of a suitable transition metal catalyst and a suitable rhodium (preferably triethylamine) and optionally additional additives such as tetrabutylammonium iodide. Union. This type of conversion is commonly referred to by the person skilled in the art as the "Heck reaction".
典型條件：DMF中的1當量的式D、10當量的乙烯基酯、2當量的四丁基碘化銨、0.2當量的Pd(dppf)Cl2 ；DMF：水：三乙胺（6.25:1:1），加熱至70℃過夜。步驟 5 Typical conditions: 1 equivalent of D in the DMF, 10 equivalents of vinyl ester, 2 equivalents of tetrabutylammonium iodide, 0.2 equivalents of Pd(dppf)Cl 2 ; DMF: water: triethylamine (6.25:1) :1), heated to 70 ° C overnight. Step 5
步驟5涉及將式E轉化為式F。該反應包括在合適的溶劑中，在合適的過渡金屬催化劑存在下，用氫源將雙鍵氫化為相應的飽和化合物。添加質子酸（如HCl或乙酸）以促進該反應可能是必需或期望的。本領域技術人員將理解，可以使用許多不同的金屬催化劑用於這種類型的反應，並且在壓力下進行這些反應可能是必需或期望的。Step 5 involves converting Formula E to Formula F. The reaction involves hydrogenating the double bond to the corresponding saturated compound with a source of hydrogen in a suitable solvent in the presence of a suitable transition metal catalyst. It may be necessary or desirable to add a protic acid such as HCl or acetic acid to facilitate the reaction. Those skilled in the art will appreciate that many different metal catalysts can be used for this type of reaction, and it may be necessary or desirable to carry out these reactions under pressure.
典型條件：在氫氣氣氛下，用碳載鈀處理式E。步驟 6 Typical conditions: Formula E is treated with palladium on carbon under a hydrogen atmosphere. Step 6
步驟6涉及將式F轉化為式G。該反應包括在合適的溶劑中，在合適的堿，如氫氧化鈉的存在下，將酯水解成相應的羧酸。Step 6 involves converting Formula F to Formula G. The reaction involves the hydrolysis of the ester to the corresponding carboxylic acid in the presence of a suitable hydrazine, such as sodium hydroxide, in a suitable solvent.
典型條件：在甲醇中用氫氧化鈉水溶液處理式F。步驟 7 Typical conditions: Formula F is treated with aqueous sodium hydroxide in methanol. Step 7
步驟7涉及將式G轉化為式H。該反應包括在醯胺鍵形成反應條件下分子內環化而形成內醯胺。本領域技術人員將理解，對於這種類型的反應可以使用許多不同的醯胺鍵形成反應條件。Step 7 involves converting Formula G to Formula H. The reaction involves intramolecular cyclization under indole bond formation reaction conditions to form the indoleamine. Those skilled in the art will appreciate that many different guanamine linkages can be used to form reaction conditions for this type of reaction.
典型條件：在二氯甲烷中，在三乙胺存在下用HATU處理式G。步驟 8 Typical conditions: Formula G is treated with HATU in the presence of triethylamine in dichloromethane. Step 8
步驟8涉及將式H轉化成通式I。反應涉及從吡唑中除去保護基團，並且精確的條件將根據保護基的性質而變化（Greene，Theodora W.和Wuts, Peter G. M. Greene's Protective Groups in Organic Synthesis，第4版.（2006）。Step 8 involves the conversion of formula H to formula I. The reaction involves the removal of the protecting group from the pyrazole, and the precise conditions will vary depending on the nature of the protecting group (Greene, Theodora W. and Wuts, Peter G. M. Greene's Protective Groups in Organic Synthesis, 4th ed. (2006).
典型條件（PG為4-甲氧基苄基）：在70℃下用三氟乙酸處理式H過夜。步驟 9 Typical conditions (PG is 4-methoxybenzyl): H is treated with trifluoroacetic acid at 70 ° C overnight. Step 9
步驟9涉及將式H轉化為式J。該反應包括用還原劑如硼烷將醯胺還原成相應的胺。本領域技術人員將理解，許多不同的還原劑可用於這種類型的反應。步驟 10 Step 9 involves converting Formula H to Formula J. The reaction involves the reduction of the indoleamine to the corresponding amine with a reducing agent such as borane. Those skilled in the art will appreciate that many different reducing agents can be used in this type of reaction. Step 10
步驟10涉及將式J轉化為式K。反應包括從吡唑除去保護基團，並且精確的條件將取決於保護基團的性質（Greene，Theodora W.和Wuts, Peter G. M. Greene’s Protective Groups in Organic Synthesis. 第4版.（2006）。Step 10 involves converting Formula J to Formula K. The reaction involves removal of the protecting group from the pyrazole, and the precise conditions will depend on the nature of the protecting group (Greene, Theodora W. and Wuts, Peter G. M. Greene's Protective Groups in Organic Synthesis. 4th ed. (2006).
典型條件（PG為4-甲氧基苄基）：在70℃下用三氟乙酸處理式J過夜。Typical conditions (PG is 4-methoxybenzyl): Formula J is treated with trifluoroacetic acid at 70 ° C overnight.
方案 2 ： 
步驟11描述了將式D轉化成式L，其中X和PG如前定義。該反應涉及在合適的溶劑中在過渡金屬催化劑的存在下，式D中的鹵化物與芳基或雜芳基硼酸或酯的交叉偶聯。反應通常在用熱的方式或微波加熱所提升的溫度下進行。通常向反應混合物中加入無機堿（如碳酸鈉）。對於本領域技術人員來說，這種類型的轉化已知為“Suzuki偶聯”。Step 11 describes the conversion of Formula D to Formula L, where X and PG are as defined above. This reaction involves the cross-coupling of a halide of formula D with an aryl or heteroaryl boronic acid or ester in the presence of a transition metal catalyst in a suitable solvent. The reaction is usually carried out at a temperature which is elevated by heat or by microwave heating. Inorganic hydrazine (such as sodium carbonate) is usually added to the reaction mixture. This type of transformation is known to those skilled in the art as "Suzuki coupling".
典型條件：1,4-二氧六環中的1當量的式D、0.09當量的Pd(dppf)2 Cl2 、1.5當量的硼酸（或硼酸酯）、3.5當量的2 M碳酸鈉水溶液在90℃下持續18小時。步驟 12 Typical conditions: 1 equivalent of formula D in 1,4-dioxane, 0.09 equivalents of Pd(dppf) 2 Cl 2 , 1.5 equivalents of boric acid (or borate), 3.5 equivalents of 2 M aqueous sodium carbonate solution It lasts for 18 hours at 90 °C. Step 12
步驟12涉及將式L轉化為式M。該反應包括在合適的溶劑中，在合適的堿，如氫氧化鈉的存在下，用水將酯水解成相應的羧酸。Step 12 involves converting Formula L to Formula M. The reaction involves the hydrolysis of the ester to the corresponding carboxylic acid with water in the presence of a suitable hydrazine, such as sodium hydroxide, in a suitable solvent.
典型條件：在甲醇中，用氫氧化鈉水溶液處理式L。步驟 13 Typical conditions: Formula L is treated with aqueous sodium hydroxide in methanol. Step 13
步驟13涉及將式M轉化為式N。該反應涉及在醯胺鍵形成反應條件下分子內環化以形成內醯胺。本領域技術人員將理解，對於這種類型的反應可以使用許多不同的醯胺鍵形成反應條件。Step 13 involves converting Formula M to Formula N. The reaction involves intramolecular cyclization under the conditions of the indole bond formation reaction to form the indoleamine. Those skilled in the art will appreciate that many different guanamine linkages can be used to form reaction conditions for this type of reaction.
典型條件：在二氯甲烷中，在三乙胺存在下用HATU處理式M。步驟 14 Typical conditions: Formula M is treated with HATU in the presence of triethylamine in dichloromethane. Step 14
步驟14涉及將式N轉化為式O。該反應涉及從吡唑除去保護基團，並且精確的條件將根據保護基的性質而變化（Greene，Theodora W.和Wuts, Peter G. M. Greene's Protective Groups in Organic Synthesis，第4版（2006）。Step 14 involves converting Formula N to Formula O. This reaction involves the removal of the protecting group from the pyrazole, and the precise conditions will vary depending on the nature of the protecting group (Greene, Theodora W. and Wuts, Peter G. M. Greene's Protective Groups in Organic Synthesis, 4th Ed. (2006).
典型條件（PG為4-甲氧基苄基）：在70℃下，用三氯乙酸處理式N過夜。步驟 15 Typical conditions (PG is 4-methoxybenzyl): The formula N is treated with trichloroacetic acid overnight at 70 °C. Step 15
步驟15涉及將式N轉化為式P。該反應包括用還原劑如硼烷將醯胺還原成相應的胺。本領域技術人員將理解，許多不同的還原劑可用於這種類型的反應。步驟 16 Step 15 involves converting Formula N to Formula P. The reaction involves the reduction of the indoleamine to the corresponding amine with a reducing agent such as borane. Those skilled in the art will appreciate that many different reducing agents can be used in this type of reaction. Step 16
步驟16涉及將式P轉化為式Q。該反應包括從吡唑除去保護基團，並且精確的條件將取決於保護基團的性質（Greene，Theodora W.和Wuts, Peter G. M. Greene's Protective Groups in Organic Synthesis. 第4版.（2006）。Step 16 involves converting Formula P to Formula Q. This reaction involves the removal of the protecting group from the pyrazole, and the precise conditions will depend on the nature of the protecting group (Greene, Theodora W. and Wuts, Peter G. M. Greene's Protective Groups in Organic Synthesis. 4th ed. (2006).
典型條件（PG為4-甲氧基苄基）：在70℃下，用三氟乙酸處理式P過夜。Typical conditions (PG is 4-methoxybenzyl): Formula P was treated with trifluoroacetic acid overnight at 70 °C.
通過以下非限制性實施例進一步描述本發明。實施例 合成化合物的常規程式 色譜法 The invention is further described by the following non-limiting examples. EXAMPLES Conventional Chromatography of Synthetic Compounds
使用由Agela Technologies製造的設備進行高壓液相色譜法，並通過多波長UV檢測器進行監測。用於分離過程的典型流動相是PE/EA、DCM/MeOH或水/MeCN。本領域技術人員將理解，改變每種具體化合物的條件可能是必須的或希望的，例如通過在開始或結束時改變溶劑組成，改變溶劑或緩衝液，改變執行時間，改變流動速率和/或色譜柱。分析方法 High pressure liquid chromatography was performed using equipment manufactured by Agela Technologies and monitored by a multi-wavelength UV detector. A typical mobile phase for the separation process is PE/EA, DCM/MeOH or water/MeCN. Those skilled in the art will appreciate that it may be necessary or desirable to vary the conditions of each particular compound, such as by changing the solvent composition at the beginning or at the end, changing the solvent or buffer, changing the execution time, changing the flow rate and/or chromatography. column. Analytical method
在室溫下在該溶劑中，使用Bruker AV 400光譜儀進行1 H核磁共振（NMR）光譜，除非另有說明。在所有情況下，NMR資料與所提出的結構一致。使用用於指定主峰的常規縮寫，以份每百萬計給出特有的化學位移（δ）：例如，s，單峰；d，二重峰；t，三重峰；q，四重峰；dd，雙二重峰；br，寬。使用Agilent 1290 Infinity/6460 triple Quad LCMS記錄質譜。當使用薄層色譜（TLC）時，它是指矽膠TLC。製備化合物 1 H nuclear magnetic resonance (NMR) spectra were carried out in this solvent at room temperature using a Bruker AV 400 spectrometer unless otherwise stated. In all cases, the NMR data was consistent with the proposed structure. Using a conventional abbreviation for specifying the main peak, a unique chemical shift (δ) is given in parts per million: for example, s, singlet; d, doublet; t, triplet; q, quartet; dd , double doublet; br, wide. Mass spectra were recorded using an Agilent 1290 Infinity/6460 triple Quad LCMS. When thin layer chromatography (TLC) is used, it refers to silicone TLC. Preparation of compounds
在沒有描述製備起始原料的情況下，這些起始原料是可以通過商業管道購買的，文獻中已知的，或者由本領域技術人員使用標準程式易於可獲得的。在說明通過類似於先前的實施例或中間體而製備化合物的情況下，本領域技術人員將理解，可以改變每個特定反應的反應時間、試劑的當量數和溫度，並且採用不同的後處理或純化技術可能是必須的或期望的。實施例 1 
合成 4- 氯 -3- 碘 -1H
- 吡唑並 [4,3-c
] 吡啶（中間體 1 ）： 
合成1-(4-甲氧基苄基)-4-氯-3-碘-1H
-吡唑並[4,3-c
]吡啶（中間體2）：
合成 1-(4- 甲氧基苄基 )-3- 碘 -N- 甲基 -1H
- 吡唑並 [4,3-c
] 吡啶 -4- 胺（中間體 3 ）： 
合成 (E)-3-(4-( 甲基氨基 )-1-(4- 甲氧基苄基 )-1H
- 吡唑並 [4,3-c
] 吡啶 -3- 基 ) 丙烯酸甲酯（中間體 4 ）： 
合成 3-(4-( 甲基氨基 )-1-(4- 甲氧基苄基 )-1H
- 吡唑並 [4,3-c
] 吡啶 -3- 基 ) 丙酸甲酯（中間體 5 ）： 
合成 3-(4-( 甲基氨基 )-1-(4- 甲氧基苄基 )-1H
- 吡唑並 [4,3-c
] 吡啶 -3- 基 )- 丙酸（中間體 6 ）： 
合成 6- 甲基 -2-(4- 甲氧基苄基 )-2,6,8,9- 四氫 -7H
-1,2,5,6- 四氮雜苯並 [cd
] 薁 -7- 酮（中間體 7 ）： 
合成 6- 甲基 -2,6,8,9- 四氫 -7H
-1,2,5,6- 四氮雜苯並 [cd
] 薁 -7- 酮（實施例 1 ）： 
在步驟3中用合適的胺代替甲胺，類似於實施例1地製備實施例2-14
。
在步驟4中用甲基丙烯酸甲酯代替丙烯酸甲酯，類似於實施例14
地製備實施例15
。
合成6-甲基-6,7,8,9-四氫-2H
-1,2,5,6-四氮雜苯並[cd
]薁（實施例16
）：
類似於實施例16
地製備實施例17-29
，如下所示，用合適的中間體代替中間體7
，該中間體是在步驟3（實施例1）中用適當的胺代替甲胺製備的。
類似於實施例16
地製備實施例30
，如下所示，用適當的中間體代替中間體7
，而該中間體是通過在步驟3（實施例1）中用適當的胺代替甲胺，並在步驟4（實施例1）中用甲基丙烯酸甲酯代替丙烯酸甲酯而製備的。
合成2-(4-(甲基氨基)-1-(4-甲氧基苄基)-1H
-吡唑並[4,3-c
]吡啶-3-基)苯甲酸甲酯（中間體9）：
合成2-(4-(乙基氨基)-1-(4-甲氧基苄基)-1H
-吡唑並[4,3-c
]吡啶-3-基）苯甲酸（中間體10
）：
合成11-(4-甲氧基苄基)-4-甲基-4,11-二氫-5H
-3,4,10,11-四氮雜二苯並[cd,h
]薁-5-酮（中間體11
）：
合成4-甲基-4,11-二氫-5H
-3,4,10,11-四氮雜二苯並[cd,h
]薁-5-酮（實施例31
）：
類似於實施例31
地製備實施例32-44
，如下所示，用合適的中間體代替中間體3
，而該中間體是在步驟3（實施例1）中用適當的胺代替甲胺而製備的。
合成4-甲基-5,11-二氫-4H
-3,4,10,11-四氮雜二苯並[cd,h
]薁（實施例45
）：
據下述方案，類似於實施例45
地製備實施例46-58
。
按照以下方案，類似於實施例44
地製備實施例59,60
，在步驟1（實施例31
）中用適當的苯基硼酸代替2-(甲氧基羰基)苯基硼酸，且在步驟3（實施例1
）中用四氫-2H
-吡喃-4-胺代替甲胺。
根據下述方案，類似於實施例58
的製備實施例61,62
，用合適的中間體代替中間體11
（實施例45
），該中間體通過在步驟1（實施例31
）中用適當的苯基硼酸代替2-(甲氧基羰基)苯基硼酸，並在步驟3（實施例1
）中用四氫-2H
-吡喃-4-胺代替甲胺。
合成4-碘-1-三苯甲基-1H
-吡唑-3-羧酸乙酯（中間體16
）：
合成4-(4,4,5,5-四甲基-1,3,2-二氧雜環戊硼烷-2-基)-1-三苯甲基-1H
-吡唑-3-羧酸乙酯（中間體17
）：
合成4-(1-(4-甲氧基苄基)-4-(四氫-2H
-吡喃-4-基氨基)-1H
-吡唑並[4,3-c
]吡啶-3-基)-1-三苯甲基-1H
-吡唑-3-羧酸乙酯（中間體18
）：
合成4-(1-(4-甲氧基苄基)-4-(四氫-2H
-吡喃-4-基氨基)-1H
-吡唑並[4,3-c
]吡啶-3-基)-1-三苯甲基-1H
-吡唑-3-羧酸（中間體19
）：
合成5-(4-甲氧基苄基)-9-(四氫-2H
-吡喃-4-基)-2-三苯甲基-5,9-二氫-1,2,4,5,8,9-六氮雜苯並[cd
]環戊二烯並[h
]薁-10(2H
)-酮（中間體20
）：
合成9-(四氫-2H
-吡喃-4-基)-5,9-二氫-1,2,4,5,8,9-六氮雜苯並[cd
]環戊二烯並[h
]薁-10(2H
)-酮（實施例 65
）：
合成5-(4-甲氧基苄基)-9-(四氫-2H
-吡喃-4-基)-2-三苯甲基-5,9-二氫-1,2,4,5,8,9-六氮雜苯並[cd
]環戊二烯並[h
]薁-10(2H
)-酮（中間體21
）：
合成5-(4-甲氧基苄基)-2-甲基-9-(四氫-2H
-吡喃-4-基)-5,9-二氫-1,2,4,5,8,9-六氮雜苯並[cd
]環戊二烯並[h]薁-10(2H
)-酮（中間體22
）和5-(4-甲氧基苄基)-1-甲基-9-(四氫-2H
-吡喃-4-基)-5,9-二氫-1,2,4,5,8,9-六氮雜苯並[cd
]環戊二烯並[h
]薁-10(1H
)-酮（中間體23
）：
合成2-甲基-9-(四氫-2H
-吡喃-4-基)-5,9-二氫-1,2,4,5,8,9-六氮雜苯並[cd
]環戊二烯並[h
]薁-10(2H
)-酮（實施例 66
）和1-甲基-9-(四氫-2H
-吡喃-4-基)-5,9-二氫-1,2,4,5,8,9-六氮雜苯並[cd
]環戊二烯[h
]薁-10(1H
)-酮（實施例 67
）：
在90℃下，將密封反應釜中的中間體22 和23 （200 mg，0.45 mmol）和TFA（5 mL）的混合物攪拌16小時。減壓濃縮混合物，並使用配備反相C-18柱的快速色譜純化殘餘物，用5%至50%乙腈/水梯度洗脫，得到20 mg的終產物66 和42 mg的終產物67 。A mixture of intermediates 22 and 23 (200 mg, 0.45 mmol) and TFA (5 mL) in a sealed kettle was stirred at <RTI ID=0.0> The mixture was concentrated under reduced pressure, and the residue was purified using flash chromatography equipped with a C-18 reverse-phase column with 5% to 50% acetonitrile / water gradient, to give the final product 20 mg 42 mg 66 and 67 of the final product.
化合物 66 :1 H NMR (400 MHz, DMSO-d 6 )δ ppm 1.63 (m, 2H), 2.80 (m, 2H), 3.46 (m, 2H), 3.94 (m, 2H), 4.13 (s, 3H), 5.71 (m, 1H), 7.10 (d,J = 5.6 Hz, 1H), 7.97 (s, 1H), 8.06 (d,J = 5.6 Hz, 1H), 13.41 (s, 1H). m/z (ESI)+ : 325 [M+H]+ 。 Compound 66 : 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm 1.63 (m, 2H), 2.80 (m, 2H), 3.46 (m, 2H), 3.94 (m, 2H), 4.13 (s, 3H ), 5.71 (m, 1H), 7.10 (d, J = 5.6 Hz, 1H), 7.97 (s, 1H), 8.06 (d, J = 5.6 Hz, 1H), 13.41 (s, 1H). m/z (ESI) + : 325 [M+H] + .
化合物 67 :1 H NMR (400 MHz, DMSO-d 6 )δ ppm 1.61 (m, 2H), 2.80 (m, 2H), 3.45 (m, 2H), 3.95 (m, 5H), 5.63 (m, 1H), 7.11 (d,J = 5.6 Hz, 1H), 8.07 (d,J = 5.6 Hz, 1H), 8.35 (s, 1H), 13.37 (s, 1H). m/z (ESI)+ : 325 [M+H]+ 。實施例 68 Compound 67 : 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm 1.61 (m, 2H), 2.80 (m, 2H), 3.45 (m, 2H), 3.95 (m, 5H), 5.63 (m, 1H) ), 7.11 (d, J = 5.6 Hz, 1H), 8.07 (d, J = 5.6 Hz, 1H), 8.35 (s, 1H), 13.37 (s, 1H). m/z (ESI) + : 325 [ M+H] + . Example 68
合成吡唑硼酯： 
合成 4- 碘 -1H - 吡唑 -3- 羧酸乙酯（ 25 ） 在室溫下，將NIS（22.5 g，100 mmol）加入至1H -吡唑-3-羧酸乙酯（10.0 g，71.4 mmol）的DCM（30 mL）溶液中。將混合物攪拌24小時。然後用H2 O（50 mL）淬滅反應物，並用EtOAc（60 mL×3）萃取混合物。用飽和食鹽水（50 mL×3）洗滌合併的有機相，通過Na2 SO4 進行乾燥。過濾後，減壓濃縮濾液。通過色譜（PE：EtOAc=1:5，v/v）純化殘餘物，得到產物4-碘-1H -吡唑-3-羧酸乙酯（15.5 g，82%）。m/z (ESI)+ : 267 [M+H]+ 。步驟 2 Synthesis of 4- iodo -1 H - pyrazole- 3- carboxylic acid ethyl ester ( 25 ) NIS (22.5 g, 100 mmol) was added to 1 H -pyrazole-3-carboxylic acid ethyl ester (10.0 ) at room temperature g, 71.4 mmol) in DCM (30 mL). The mixture was stirred for 24 hours. Then treated with H 2 O (50 mL) The reaction was quenched, and the mixture was extracted with EtOAc (60 mL × 3). The combined organic phases were washed with brine (50 mL×3) and dried over Na 2 SO 4 . After filtration, the filtrate was concentrated under reduced pressure. By chromatography (PE: EtOAc = 1: 5 , v / v) and the residue was purified to give the product 4-iodo -1 H - pyrazole-3-carboxylate (15.5 g, 82%). m/z (ESI) + : 267 [M+H] + . Step 2
合成 4- 碘 -1- 異丙基 -1H - 吡唑 -5- 羧酸乙酯（ 26 ）和 4- 碘 -1- 異丙基 -1H - 吡唑 -3- 羧酸乙酯（ 27 ） 將2-碘丙烷（12.9 g，75.9 mmol）和K2 CO3 （16.0 g，116 mmol）加入至4-碘-1H -吡唑-3-羧酸乙酯（15.53 g，58.4 mmol）的DMF（150 mL）溶液中。將混合物加熱至60℃持續反應3小時。然後用水（300 mL）淬滅反應，用EtOAc（200 mL×3）萃取混合物。用飽和食鹽水（200 mL×3）洗滌合併的有機相並通過Na2 SO4 進行乾燥。過濾後，減壓濃縮有機相。通過正相柱層析（PE:EtOAc=10:1，v/v）純化殘餘物，得到4-碘-1-異丙基-1H -吡唑-5-羧酸乙酯（中間體26 ）（9.4 g，52%），m/z (ESI)+ : 309 [M+H]+ ，且利用PE:EtOAc=8:1（v/v）洗脫，得到4-碘-1-異丙基-1H -吡唑-3-羧酸乙酯（中間體27 ）（6.1 g，33%）。m/z (ESI)+ : 309 [M+H]+ 。步驟 3 Synthesis of ethyl 4- iodo- 1- isopropyl- 1 H - pyrazole- 5- carboxylate ( 26 ) and ethyl 4- iodo- 1- isopropyl- 1 H - pyrazole- 3- carboxylate ( 27 ) 2-Iodopropane (12.9 g, 75.9 mmol) and K 2 CO 3 (16.0 g, 116 mmol) were added to ethyl 4-iodo-1 H -pyrazole-3-carboxylate (15.53 g, 58.4 mmol ) in DMF (150 mL) solution. The mixture was heated to 60 ° C for 3 hours. The reaction was quenched with water (300 mL)EtOAcEtOAc The combined organic phases were washed with brine (200 mL×3) and dried over Na 2 SO 4 . After filtration, the organic phase was concentrated under reduced pressure. By normal phase column chromatography (PE: EtOAc = 10: 1 , v / v) and the residue was purified to give 4-iodo-1-isopropyl--1 H - pyrazole-5-carboxylate (Intermediate 26 (9.4 g, 52%), m/z (ESI) + : 309 [M+H] + , eluted with PE:EtOAc = 8:1 (v/v) Ethyl propyl- 1H -pyrazole-3-carboxylate (Intermediate 27 ) (6.1 g, 33%). m/z (ESI) + : 309 [M+H] + . Step 3
合成 1- 異丙基 -4-(4,4,5,5- 四甲基 -1,3,2- 二氧雜環戊硼烷 -2- 基 )-1H - 吡唑 -5- 羧酸乙酯（ 28 ） 將4,4,4',4',5,5,5',5'-八甲基-2,2'-聯(1,3,2-二氧雜環戊硼烷）（3.56 g，14.0 mmol）、KOAc（2.7 g，27.5 mmol）和Pd(dppf)2 Cl2 （300 mg）加入至4-碘-1-異丙基-1H -吡唑-5-羧酸乙酯（2.14 g，6.95 mmol）的DMSO（20 mL）溶液中。將混合物加熱至100℃持續2小時。然後用H2 O（40 mL）淬滅反應並用EtOAc（40 mL×3）萃取混合物。然後用飽和食鹽水（40 mL×3）洗滌合併的有機相，通過Na2 SO4 進行乾燥。過濾後，減壓濃縮濾液。通過快速柱層析色譜（PE:EtOAc=10:1，v/v）純化殘餘物，得到產物1-異丙基-4-(4,4,5,5-四甲基-1,3,2-二氧雜環戊硼烷-2-基)-1H -吡唑-5-羧酸乙酯（1.0 g，93%）。m/z (ESI)+ : 309 [M+H]+ 。步驟 4 Synthesis of 1- isopropyl- 4-(4,4,5,5 -tetramethyl -1,3,2- dioxaborolan- 2- yl )-1 H - pyrazole- 5- carboxylate Ethyl ester ( 28 ) will be 4,4,4',4',5,5,5',5'-octamethyl-2,2'-linked (1,3,2-dioxaboroboron) Alkyl) (3.56 g, 14.0 mmol), KOAc (2.7 g, 27.5 mmol) and Pd(dppf) 2 Cl 2 (300 mg) were added to 4-iodo-1-isopropyl-1 H -pyrazole-5- Ethyl carboxylate (2.14 g, 6.95 mmol) in DMSO (20 mL). The mixture was heated to 100 ° C for 2 hours. Then treated with H 2 O (40 mL) and the reaction was quenched mixture was extracted with EtOAc (40 mL × 3). Then with saturated brine (40 mL × 3) The combined organic phases were washed by Na 2 SO 4 dried. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography (EtOAc:EtOAc:EtOAc:EtOAc Ethyl 2-dioxaborolan-2-yl)-1 H -pyrazole-5-carboxylate (1.0 g, 93%). m/z (ESI) + : 309 [M+H] + . Step 4
合成 1- 異丙基 -4-(4,4,5,5- 四甲基 -1,3,2- 二氧雜環戊硼烷 -2- 基 )-1H - 吡唑 -3- 羧酸乙酯（ 29 ） 將4-碘-1-異丙基-1H -吡唑-3-羧酸乙酯（4.5 g，14.6 mmol）和2-異丙氧基-4,4,5,5-四甲基-1,3,2-二氧雜環戊硼烷（8.15 g，43.8 mmol）的無水THF（50 mL）溶液冷卻至-75℃，然後逐滴地滴加n-BuLi（17.5 mL，正己烷溶液，1.6N，43.8 mmol），將混合物在-75℃下攪拌2小時，隨後加入200 mL飽和NH4 Cl水溶液淬滅反應，用乙酸乙酯（100 mL×3）萃取混合物，並用50 mL飽和食鹽水洗滌合併的有機層，進行乾燥（Na2 SO4 ）並減壓濃縮。用快速色譜純化殘餘物（黃色油狀物），用0至40%EtOAc/PE梯度洗脫，得到淡黃色油狀物（2.0 g，44%）。1 H NMR (400 MHz, DMSO-d 6 )δ ppm 1.20-1.50 (m, 21H), 4.25 (q,J = 7.2 Hz, 2H), 4.51-4.65 (m, 1 H), 8.18 (s, 1 H); m/z (ESI)+ : 309 [M+H]+ 。 Synthesis of 1- isopropyl- 4-(4,4,5,5 -tetramethyl -1,3,2- dioxaborolan- 2- yl )-1 H - pyrazole- 3- carboxylate Ethyl ester ( 29 ) ethyl 4-iodo-1-isopropyl-1 H -pyrazole-3-carboxylate (4.5 g, 14.6 mmol) and 2-isopropoxy-4,4,5, A solution of 5-tetramethyl-1,3,2-dioxaborolane (8.15 g, 43.8 mmol) in dry THF (50 mL) was cooled to -75 ° C, then n-BuLi was added dropwise. 17.5 mL, n-hexane solution, 1.6N, 43.8 mmol), and the mixture was stirred for 2 hours at -75 deg.] C, followed by addition of 200 mL 4 Cl quenched with saturated aqueous NH off the reaction, the mixture was extracted with ethyl acetate (100 mL × 3) , and the combined organic layer was washed with saturated saline water, 50 mL, dried (Na 2 SO 4) and concentrated under reduced pressure. The residue was purified by flash chromatography eluting elut elut elut elut elut elut 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm 1.20-1.50 (m, 21H), 4.25 (q, J = 7.2 Hz, 2H), 4.51-4.65 (m, 1 H), 8.18 (s, 1 H); m/z (ESI) + : 309 [M+H] + .
可以類似於Can be similar 2828 和with 2929 在步驟In the steps 22 中使用碘甲烷或碘乙烷製備下面的吡唑硼酯：The following pyrazole boron esters were prepared using methyl iodide or ethyl iodide:
合成實施例Synthesis example 6868 ：: 步驟step 11
合成 3- 碘 -1-(4- 甲氧基苄基 )-N-(1- 甲基呱啶 -4- 基 )-1H - 吡唑並 [4,3-c ] 吡啶 -4- 胺（ 30 ） 在130℃下，將在50 mL圓底燒瓶中的4-氯-3-碘-1-(4-甲氧基苄基)-1H -吡唑並[4,3-c ]吡啶（3.0 g，7.5 mmol）、1-甲基呱啶-4-胺（2.57 g，22.5 mmol）和DIEA（3.87 g，30 mmol）的混合物攪拌過夜。隨後減壓濃縮反應物，並用正相柱層析純化殘餘物，用DCM/MeOH梯度洗脫，得到棕色固體3-碘-1-(4-甲氧基苄基)-N-(1-甲基呱啶-4-基)-1H -吡唑並[4,3-c ]吡啶-4-胺（2.7 g，75%）。m/z (ESI)+ : 478 [M+H]+ 。步驟 2 Synthesis of 3- iodo- 1-(4 -methoxybenzyl )-N-(1 -methylacridin- 4 -yl )-1 H - pyrazolo [4,3- c ] pyridin- 4- amine ( 30 ) 4-Chloro-3-iodo-1-(4-methoxybenzyl)-1 H -pyrazolo[4,3- c ] in a 50 mL round bottom flask at 130 °C A mixture of pyridine (3.0 g, 7.5 mmol), 1-methyl acridin-4-amine (2.57 g, 22.5 mmol) and DIEA (3.87 g, 30 mmol) was stirred overnight. The reaction was then concentrated under reduced EtOAcqqqqHHHHHHHHHHHHHHHHHHHHH Acridine-4-yl)-1 H -pyrazolo[4,3- c ]pyridin-4-amine (2.7 g, 75%). m/z (ESI) + : 478 [M+H] + . Step 2
合成 1- 異丙基 -4-(1-(4- 甲氧基苄基 )-4-((1- 甲基呱啶 -4- 基 ) 氨基 )-1H - 吡唑並 [4,3-c ] 吡啶 -3- 基 )-1H - 吡唑 -5- 羧酸乙酯（ 31 ） 在N2 保護下，將3-碘-1-(4-甲氧基苄基)-N-(1-甲基呱啶-4-基)-1H -吡唑並[4,3-c ]吡啶-4-胺（464 mg，0.97 mmol）、1-異丙基-4-(4,4,5,5-四甲基-1,3,2-二氧雜環戊硼烷-2-基)-1H -吡唑-5-羧酸乙酯（300 mg，0.97 mmol），Pd(dppf)Cl2 （80 mg，0.097 mmol）和2N Na2 CO3 （1 mL，2 mmol）的1,4-二氧六環（20 mL）溶液加熱至90℃並攪拌過夜。隨後用EtOAc（100 mL）稀釋混合物，用水（30 mL×2）、飽和食鹽水（30 mL）洗滌並通過Na2 SO4 進行乾燥。過濾有機相並濃縮濾液，得到殘餘物，將該殘餘物通過矽膠色譜純化，用DCM/MeOH=10/1（v/v）洗脫，得到黃色油狀物1-異丙基-4-(1-(4-甲氧基苄基)-4-((1-甲基呱啶-4-基)氨基)-1H -吡唑並[4,3-c ]吡啶-3-基)-1H -吡唑-5-羧酸乙酯（300 mg，58%）。m/z (ESI)+ : 532 [M+H]+ 。步驟 3 Synthesis of 1- isopropyl- 4-(1-(4 -methoxybenzyl )-4-((1 -methylacridin- 4 -yl ) amino )-1 H - pyrazolo [4,3 - c ] Pyridin- 3 -yl )-1 H - pyrazole- 5- carboxylic acid ethyl ester ( 31 ) 3-iodo-1-(4-methoxybenzyl)-N- under N 2 protection (1-methylacridin-4-yl)-1 H -pyrazolo[4,3- c ]pyridin-4-amine (464 mg, 0.97 mmol), 1-isopropyl-4-(4, Ethyl 4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1 H -pyrazole-5-carboxylate (300 mg, 0.97 mmol), Pd A solution of (dppf) Cl 2 (80 mg, 0.097 mmol) and 2N Na 2 CO 3 (1 mL, 2 mmol) in 1,4-dioxane (20 mL) was warmed to 90 ° C and stirred overnight. The mixture was then diluted with water (30 mL × 2) with EtOAc (100 mL), saturated brine (30 mL) was washed and dried over Na 2 SO 4. The organic phase was filtered, and EtOAc was evaporated eluted elut elut elut elut elut elut elut elut 1-(4-Methoxybenzyl)-4-((1-methylacridin-4-yl)amino)-1 H -pyrazolo[4,3- c ]pyridin-3-yl)- 1 H -pyrazole-5-carboxylic acid ethyl ester (300 mg, 58%). m/z (ESI) + : 532 [M+H] + . Step 3
合成 (1- 異丙基 -4-(1-(4- 甲氧基苄基 )-4 -((1- 甲基呱啶 -4- 基 ) 氨基 )-1H - 吡唑並 [4,3-c ] 吡啶 -3- 基 ) -1H - 吡唑 -5- 基 ) 甲醇（ 32 ） 在0o C下，將LiAlH4 （182 mg，4.79 mmol）分批加入至1-異丙基-4-(1-(4-甲氧基苄基)-4-((1-甲基呱啶-4-基)氨基)-1H -吡唑並[4,3-c ]吡啶-3-基)-1H -吡唑-5-羧酸乙酯（510 mg，0.96 mmol）的無水THF（20 mL）溶液中。將混合物在室溫下攪拌1小時。隨後用水（5 mL）和EtOAc（100 mL）淬滅反應。過濾懸浮液，分離有機相並用飽和食鹽水（15 mL）洗滌，隨後通過Na2 SO4 進行乾燥。過濾有機相，並減壓濃縮濾液。通過矽膠色譜，以DCM/MeOH=6/1（v/v）洗脫來純化殘餘物，得到白色固體(1-異丙基-4-(1-(4-甲氧基苄基)-4-((1-甲基呱啶-4-基)氨基)-1H-吡唑並[4,3-c ]吡啶-3-基)-1H-吡唑-5-基)甲醇（300 mg，64%）。m/z (ESI)+ : 490 [M+H]+ 。步驟 4 Synthesis of 1- isopropyl- 4-(1-(4 -methoxybenzyl )-4 - ((1 -methylacridin- 4 -yl ) amino )-1 H - pyrazolo [4, 3- c] pyridin-3-yl) - 1 H - pyrazol-5-yl) methanol (32) at 0 o C, the LiAlH 4 (182 mg, 4.79 mmol ) was added portionwise to l-isopropyl- 4-(1-(4-methoxybenzyl)-4-((1-methylacridin-4-yl)amino)-1 H -pyrazolo[4,3- c ]pyridine-3 To a solution of ethyl- 1H -pyrazole-5-carboxylate (510 mg, 0.96 mmol) in anhydrous THF (20 mL). The mixture was stirred at room temperature for 1 hour. The reaction was then quenched with water (5 mL)EtOAcEtOAcEtOAc The suspension was filtered, separated and the organic phase was washed with saturated brine (15 mL), then dried over Na 2 SO 4. The organic phase was filtered and the filtrate was concentrated evaporated. The residue was purified by EtOAc EtOAc EtOAc elut elut elut elut -((1-methylacridin-4-yl)amino)-1H-pyrazolo[4,3- c ]pyridin-3-yl)-1H-pyrazol-5-yl)methanol (300 mg, 64%). m/z (ESI) + : 490 [M+H] + . Step 4
合成 1- 異丙基 -5-(4- 甲氧基苄基 )-9-(1- 甲基呱啶 -4- 基 )-1,5,9,10- 四氫 -1,2,4,5,8,9- 六氮雜苯並 [cd ] 環戊二烯並 [h ] 薁（ 33 ） 在0℃在N2 下，將甲磺醯氯（144 mg，1.22 mmol）逐滴地加入至(1-異丙基-4-(1-(4-甲氧基苄基)-4-((1-甲基呱啶-4-基)氨基)-1H -吡唑並[4,3-c ]吡啶-3-基)-1H -吡唑-5-基)甲醇（300 mg，0.61 mmol）和Et3 N（185 mg，1.84 mmol）的THF（50 mL）溶液中。隨後將反應在室溫下攪拌0.5小時，TLC顯示起始原料消失，隨後加入t- BuOK（206 mg，1.84 mmol）。將反應在N2 保護下加熱至60℃攪拌1小時，隨後用水（10 mL）淬滅反應並用EtOAc（100 mL）萃取，用飽和食鹽水（30 mL）洗滌有機相，通過Na2 SO4 進行乾燥。過濾有機相並濃縮濾液，得到殘餘物，通過以DCM/MeOH=6/1（v/v）洗脫的矽膠色譜純化該殘餘物，得到無色油狀物1-異丙基-5-(4-甲氧基苄基)-9-(1-甲基呱啶-4-基)-1,5,9,10-四氫-1,2,4,5,8,9-六氮雜苯並[cd ]環戊二烯並[h ]薁（200 mg，69%）。m/z (ESI)+ : 472 [M+H]+ 。步驟 5 Synthesis of 1- isopropyl- 5-(4 -methoxybenzyl )-9-(1 -methylacridin- 4 -yl )-1,5,9,10 -tetrahydro- 1,2,4 ,5,8,9 - hexaazabenzo [ cd ] cyclopenta [ h ] pyrene( 33 ) dropwise at 0 ° C under N 2 with methanesulfonate chloride (144 mg, 1.22 mmol) Add to (1-isopropyl-4-(1-(4-methoxybenzyl)-4-((1-methylacridin-4-yl)amino)-1 H -pyrazolo[4 , 3- c] pyridin-3-yl) -1 H - pyrazol-5-yl) methanol (300 mg, 0.61 mmol) and Et 3 N (185 mg, 1.84 mmol) in THF (50 mL) solution. The reaction was then stirred at room temperature for 0.5 h. TLC showed the starting material disappeared and then t- BuOK (206 mg, 1.84 mmol). The reaction under N 2 was heated to 60 deg.] C stirred for 1 hour, then washed with water (10 mL) quenched the reaction and extracted with EtOAc (100 mL), washed with saturated brine (30 mL) The organic phase was washed by Na 2 SO 4 dry. The organic phase was filtered, and the filtrate was evaporated tolululululululululululululululululululululu -methoxybenzyl)-9-(1-methylacridin-4-yl)-1,5,9,10-tetrahydro-1,2,4,5,8,9-hexazabenzene And [ cd ]cyclopentadienyl [ h ] 薁 (200 mg, 69%). m/z (ESI) + : 472 [M+H] + . Step 5
合成 1- 異丙基 -9-(1- 甲基呱啶 -4- 基 )-1,5,9,10- 四氫 -1,2,4,5,8,9- 六氮雜苯並 [cd ] 環戊二烯並 [h ] 薁（實施例 68 ） 在70℃下，將1-異丙基-5-(4-甲氧基苄基)-9-(1-甲基呱啶-4-基)-1,5,9,10-四氫-1,2,4,5,8,9-六氮雜苯並[cd ]環戊二烯並[h ]薁（200 mg，0.42 mmol）在TFA（5 mL）中的混合物攪拌過夜，減壓濃縮，通過製備型HPLC純化所得殘餘物，合併所收集的洗脫液，用飽和的NaHCO3 水溶液調節至pH=8，並用EtOAc（10 mL×3）萃取，通過Na2 SO4 乾燥有機相並進行過濾，減壓濃縮濾液，得到白色固體1-異丙基-9-(1-甲基呱啶-4-基)-1,5,9,10-四氫-1,2,4,5,8,9-六氮雜苯並[cd ]環戊二烯並[h ]薁（110 mg，74%）。1 H NMR (400 MHz, DMSO-d 6 )δ ppm 1.44 (d,J = 6.0 Hz, 6H), 1.63-1.71 (m, 2H), 1.91-2.12 (m, 4H), 2.22-2.32 (m, 3H), 2.92 (s, 2H), 4.47 (s, 2H), 4.62 (m, 1H), 4.93-4.96(m, 1H), 6.7，9 (d,J = 6.0 Hz, 1H), 7.78-7.81 (m, 2H), 12.95 (br s, 1H); m/z (ESI)+ : 352 [M+H]+ 。實施例 69-88 Synthesis of 1-isopropyl-9- (1-methyl-piperidin-4-yl) -1,5,9,10- tetrahydro-benzo -1,2,4,5,8,9- hexaaza [ cd ] cyclopentadien [ h ] pyrene (Example 68 ) 1-isopropyl-5-(4-methoxybenzyl)-9-(1-methylacridine ) at 70 ° C 4-yl)-1,5,9,10-tetrahydro-1,2,4,5,8,9-hexaazabenzo[ cd ]cyclopenta[ h ]pyrene (200 mg, 0.42 mmol) in a mixture of TFA (5 mL) was stirred overnight, concentrated under reduced pressure, the collected purified by preparative HPLC resulting residue was combined eluate was adjusted with saturated aqueous NaHCO 3 to pH = 8, and extracted with EtOAc (10 mL × 3), the organic phase was dried over Na 2 SO 4 and filtered, and the filtrate was evaporated to give a white solid, 1-isopropyl-9-(l-methyl apyridin-4-yl)-1 , 5,9,10-tetrahydro-1,2,4,5,8,9-hexazabenzo[ cd ]cyclopenta[ h ]pyrene (110 mg, 74%). 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm 1.44 (d, J = 6.0 Hz, 6H), 1.63-1.71 (m, 2H), 1.91-2.12 (m, 4H), 2.22-2.32 (m, 3H), 2.92 (s, 2H), 4.47 (s, 2H), 4.62 (m, 1H), 4.93-4.96 (m, 1H), 6.7,9 (d, J = 6.0 Hz, 1H), 7.78-7.81 (m, 2H), 12.95 (br s, 1H); m/z (ESI) + : 352 [M+H] + . Example 69-88
類似於實施例68
，在步驟1中使用合適的胺以及在步驟2中使用適當的吡唑硼酯而製備實施例69-88
。對於實施例69
和76
，合適的吡唑硼酯是實施例65
步驟2中的中間體17
。
合成 4- 碘 -6-( 三氟甲基 ) 煙酸（ 34 ） 在3000 mL的3頸圓底燒瓶中，氮氣保護，將2,2,6,6-四甲基呱啶（34.5 g，141 mmol）的無水THF（600 mL）溶液降溫-78℃，維持-78℃，將n -BuLi（98 mL，2.4M己烷溶液）加入並攪拌反應0.5小時，後將6-三氟甲基煙酸（15 g，78 mmol）的無水THF（50 mL）溶液逐滴地加入，並在-78℃下將混合物攪拌2.5小時。隨後緩慢加入I2 （29.3 g，117 mmol）的無水THF（50 mL）溶液，並同樣在-78℃下攪拌反應1小時。隨後將100 mL飽和NH4 Cl水溶液加入淬滅反應，通過1N HCl將反應液的pH值調節至4〜5，隨後用DCM（400 mL×3）萃取混合物， Na2 SO4 進行乾燥，過濾並減壓濃縮，得到殘餘物，通過在正相矽膠柱純化（PE/EA/AcOH，1:10:0.01，v/v/v），得到黃色固體化合物34 （10 g，40%）。m/z (ESI)- : 316 [M-H]- 。步驟 2 Synthesis of 4- iodo -6-( trifluoromethyl ) nicotinic acid ( 34 ) in a 3000 mL 3-neck round bottom flask with nitrogen protection, 2,2,6,6-tetramethylacridine (34.5 g, 141 mmol) of anhydrous THF (600 mL) was cooled to -78 ° C, maintained at -78 ° C, n- BuLi (98 mL, 2.4 M solution in hexane) was added and stirred for 0.5 hour, then 6-trifluoromethyl A solution of nicotinic acid (15 g, 78 mmol) in dry THF (50 mL) was added dropwise and the mixture was stirred at -78 ° C for 2.5 hr. Then, a solution of I 2 (29.3 g, 117 mmol) in anhydrous THF (50 mL) was slowly added, and the mixture was stirred at -78 ° C for 1 hour. Subsequently, 100 mL of a saturated aqueous solution of NH 4 Cl was added to the quenching reaction, and the pH of the reaction mixture was adjusted to 4 to 5 by 1N HCl, then the mixture was extracted with DCM (400 mL×3), dried over Na 2 SO 4 and filtered. concentrated under reduced pressure to give a residue, purified by normal phase (PE / EA / AcOH, 1 : 10: 0.01, v / v / v) silica gel column to give a yellow solid compound 34 (10 g, 40%) . m/z (ESI) - : 316 [MH] - . Step 2
合成 4- 碘 -6-( 三氟甲基 ) 煙酸甲酯（ 35 ） 在室溫下，將煙酸衍生物34 （10 g，31 mmol），K2 CO3 （8.7 g，63 mmol）和MeI（5.37 g，37.9 mmol）在DMF（25 mL）中的混合物攪拌過夜。隨後向反應混合物中加入H2 O（300 mL）。過濾混合物並用水（20 mL）洗滌濾餅，在空氣中乾燥，得到黃色固體化合物35 （10 g，粗品）。m/z (ESI)+ : 332 [M+H]+ 。步驟 3 Synthesis of methyl 4- iodo -6-( trifluoromethyl ) nicotinate ( 35 ) at room temperature with nicotinic acid derivative 34 (10 g, 31 mmol), K 2 CO 3 (8.7 g, 63 mmol) A mixture of MeI (5.37 g, 37.9 mmol) in DMF (25 mL) was stirred overnight. H 2 O (300 mL) was then added to the reaction mixture. The mixture was filtered and washed with water (20 mL) was washed cake was dried in air, to give a yellow solid compound 35 (10 g, crude). m/z (ESI) + : 332 [M+H] + . Step 3
合成 4-(4,4,5,5- 四甲基 -1,3,2- 二氧雜環戊硼烷 -2- 基 )-6-( 三氟甲基 ) 煙酸甲酯（ 36 ） 在N2 保護下，將碘化物35 （10.0 g，30.0 mmol）、雙(頻哪醇合)二硼（11.5 g，45.3 mmol）、KOAc（5.9 g，60.4 mmol）和Pd(dppf)2 Cl2 （3.31 g，4.53 mmol）混合於1,4-二氧六環（100 mL）中，升溫至95℃並維持該溫度持續攪拌18小時。後降溫濃縮，殘餘物在於EtOAc（300 mL）和水（80 mL）之間分液。用水（200 mL×2）洗滌有機層，用無水Na2 SO4 乾燥，過濾並減壓濃縮。通過正相矽膠柱層析（PE/EA，v/v，20:1至10:1梯度）純化殘餘物，得到黃色固體的產物（2.2 g）。m/z (ESI)+ : 250 [M+H]+ 。步驟 4 Synthesis of methyl 4-(4,4,5,5 -tetramethyl -1,3,2- dioxaborolan- 2- yl )-6-( trifluoromethyl ) nicotinate ( 36 ) Iodide 35 (10.0 g, 30.0 mmol), bis(pinacol) diboron (11.5 g, 45.3 mmol), KOAc (5.9 g, 60.4 mmol) and Pd(dppf) 2 Cl under N 2 protection 2 (3.31 g, 4.53 mmol) was mixed in 1,4-dioxane (100 mL), warmed to 95 ° C and maintained at this temperature and stirred for 18 hours. After concentrating, the residue was partitioned between EtOAc (300 mL) and water (EtOAc). The organic layer (200 mL × 2) washed, dried over anhydrous Na 2 SO 4, filtered, and concentrated under reduced pressure with water. The residue was purified by EtOAc EtOAc EtOAc (EtOAc) m/z (ESI) + : 250 [M+H] + . Step 4
合成 4-(1-(4- 甲氧基苄基 )-4 -(( 四氫 -2H - 吡喃 -4- 基 ) 氨基 )-1H - 吡唑並 [4,3-c ] 吡啶 -3- 基 )-6-( 三氟甲基 ) 煙酸（ 37 ） 用N2 將化合物3a （1.23 g，2.66 mmol）、化合物36 （2.2 g，6.6 mmol）和K2 CO3 （733 mg，5.32 mmol）的1,4-二氧六環/H2 O（4:1，v/v，20 mL）溶液脫氣。隨後，向上述溶液中加入PPh3 （175 mg，0.67 mmol）和Pd(PPh3 )4 （185 mg）。在氮氣保護下，將混合物在100℃下攪拌18小時。減壓濃縮反應混合物，得到殘餘物，通過正相矽膠柱層析純化（DCM/MeOH，v/v，5:1至2:1梯度洗脫），得到化合物37 ，為黃色固體（1.0 g，69%）。m/z (ESI)+ : 528 [M+H]+ 。步驟 5 Synthesis of 4-(1-(4 -methoxybenzyl )-4 - (( tetrahydro- 2 H - pyran- 4 -yl ) amino )-1 H - pyrazolo [4,3- c ] pyridine 3 -yl )-6-( trifluoromethyl ) nicotinic acid ( 37 ) Compound 3a (1.23 g, 2.66 mmol), compound 36 (2.2 g, 6.6 mmol) and K 2 CO 3 (733 mg ) with N 2 , 5.32 mmol) of 1,4-dioxane/H 2 O (4:1, v/v, 20 mL) solution was degassed. Subsequently, PPh 3 (175 mg, 0.67 mmol) and Pd(PPh 3 ) 4 (185 mg) were added to the above solution. The mixture was stirred at 100 ° C for 18 hours under a nitrogen atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue, purified by normal phase silica gel column chromatography (DCM / MeOH, v / v , 5: 1 to 2: 1 gradient elution) to give compound 37 as a yellow solid (1.0 g, 69%). m/z (ESI) + : 528 [M+H] + . Step 5
合成 (4-(1-(4- 甲氧基苄基 )-4 -(( 四氫 -2H - 吡喃 -4- 基 ) 氨基 )-1H - 吡唑並 [4,3-c ] 吡啶 -3- 基 )-6 -( 三氟甲基 ) 吡啶 -3- 基 ) 甲醇（ 38 ） 在0℃下，將中間體37 （1 g，1.9 mmol），AlCl3 （234 mg，1.9 mmol）和LiAlH4 （261 mg，7.86 mmol）在無水THF（40 mL）中的混合物攪拌1小時。依次地將H2 O（0.26 mL）、15%NaOH（水溶液，0.26 mL）和H2 O（0.8 mL）加入至混合物中。過濾混合物並減壓濃縮濾液，正相矽膠柱層析純化殘餘物（PE/EA，v/v，5:1至2:1梯度洗脫），得到為黃色油狀物的產物（240 mg，24.6%）。m/z (ESI)+ : 514 [M+H]+ 。步驟 6 Synthesis of 4-(1-(4 -methoxybenzyl )-4 - (( tetrahydro- 2 H - pyran- 4 -yl ) amino )-1 H - pyrazolo [4,3- c ] pyridin-3-yl) -6 - (trifluoromethyl) pyridin-3-yl) methanol (38) at 0 ℃, intermediate 37 (1 g, 1.9 mmol) , AlCl 3 (234 mg, 1.9 mmol And a mixture of LiAlH 4 (261 mg, 7.86 mmol) in dry THF (40 mL). H 2 O (0.26 mL), 15% NaOH (aqueous solution, 0.26 mL) and H 2 O (0.8 mL) were sequentially added to the mixture. The mixture was filtered, and the filtrate was evaporated.jjjjjjjjjjjjjjjjjjjjj 24.6%). m/z (ESI) + : 514 [M+H] + . Step 6
合成 11-(4- 甲氧基苄基 )-4-( 四氫 -2H- 吡喃 -4- 基 )-8-( 三氟甲基 )-5,11- 二氫 -4H-3,4,7,10,11- 五氮雜二苯並 [cd ,h ] 薁（ 39 ） 在0℃，將MsCl（112 mg，0.97 mmol）逐滴地加入至中間體38 （250 mg，0.49 mmol）和TEA（198 mg，1.95 mmol）的無水THF（20 mL）溶液中。在0℃下攪拌0.5小時後，加入t- BuOK（219 mg，1.95 mmol）。隨後在55℃下攪拌反應1.5小時，減壓濃縮，得到殘餘物，正相矽膠柱層析純化殘餘物（PE/EA，1:1，v/v），得到為黃色固體的化合物39 （230 mg，95%）。m/z (ESI)+ : 496 [M+H]+ 。步驟 7 Synthesis of 11-(4 -methoxybenzyl )-4-( tetrahydro -2H- pyran- 4 -yl )-8-( trifluoromethyl )-5,11 -dihydro- 4H-3,4 ,7,10,11 - pentazadibenzo [ cd , h ] 薁( 39 ) MsCl (112 mg, 0.97 mmol) was added dropwise to Intermediate 38 (250 mg, 0.49 mmol) at 0 °C And a solution of TEA (198 mg, 1.95 mmol) in dry THF (20 mL). After stirring at 0 °C for 0.5 h, t- BuOK (219 mg, 1.95 mmol) was added. Followed by stirring at 55 ℃ 1.5 h, concentrated under reduced pressure to give the residue was purified by normal phase silica gel column chromatography the residue (PE / EA, 1: 1 , v / v), to give compound 39 as a yellow solid (230 Mg, 95%). m/z (ESI) + : 496 [M+H] + . Step 7
合成 4-( 四氫 -2H - 吡喃 -4- 基 )-8-( 三氟甲基 )-5,11- 二氫 -4H -3,4,7,10,11- 五氮雜二苯並 [cd ,h ] 薁（實施例 89 ） 化合物39 （230 mg，0.46 mmol）在微波反應管中溶於TFA（3 mL），後微波輔助加熱至105℃反應攪拌1小時，後減壓濃縮，殘餘物通過製備型HPLC純化，得到白色固體（55 mg，33.1%）。1 H-NMR (400 MHz, DMSO-d6 )δ ppm 1.76 (d,J = 10.6 Hz, 2H), 2.18 (dt,J = 11.5, 7.7 Hz, 2H), 3.47 (t,J = 11.4 Hz, 2H), 4.04 (dd,J = 11.1, 3.6 Hz, 2H), 4.41 (t,J = 10.2 Hz, 1H), 4.89 (s, 2H), 7.24 (d,J = 6.8 Hz, 1H), 7.86 (d,J = 6.9 Hz, 1H), 8.27 (s, 1H), 9.19 (s, 1H), 14.70 (s, 1H);19 F-NMR (376 MHz, DMSO-d6 )δ ppm -66.72, -74.41; m/z (ESI)+ : 376 [M+H]+ .。實施例 90-92 Synthesis of 4-( tetrahydro- 2 H - pyran- 4 -yl )-8-( trifluoromethyl )-5,11 -dihydro- 4 H -3,4,7,10,11 - pentaza Dibenzo [ cd , h ] 薁 (Example 89 ) Compound 39 (230 mg, 0.46 mmol) was dissolved in TFA (3 mL) in a microwave reaction tube, and then microwave-assistedly heated to 105 ° C for 1 hour, then reduced Concentration by pressure, the residue was purified by EtOAc EtOAc EtOAc 1 H-NMR (400 MHz, DMSO- d 6 ) δ ppm 1.76 (d, J = 10.6 Hz, 2H), 2.18 (dt, J = 11.5, 7.7 Hz, 2H), 3.47 (t, J = 11.4 Hz, 2H), 4.04 (dd, J = 11.1, 3.6 Hz, 2H), 4.41 (t, J = 10.2 Hz, 1H), 4.89 (s, 2H), 7.24 (d, J = 6.8 Hz, 1H), 7.86 ( d, J = 6.9 Hz, 1H), 8.27 (s, 1H), 9.19 (s, 1H), 14.70 (s, 1H); 19 F-NMR (376 MHz, DMSO- d 6 ) δ ppm -66.72, - 74.41; m/z (ESI) + : 376 [M+H] + . Example 90-92
在步驟1中使用合適的吡啶，類似於實施例89
地製備實施例90-92
。
合成 2 -((1-(4- 甲氧基苄基 )-4 -(( 四氫 -2H - 吡喃 -4- 基 ) 氨基 )-1H - 吡唑並 [4,3-c ] 吡啶 -3- 基 )( 甲基 ) 氨基 ) 乙醇（ 40 ） 在100℃下，將3-碘-1-(4-甲氧基苄基)-N-(四氫-2H -吡喃-4-基)-1H -吡唑並[4,3-c ]吡啶-4-胺（1.5 g，3.23 mmol）、2-(甲基氨基)乙醇（1.98 g，32.3 mmol）、碘化亞銅（123 mg，0.65 mmol）、L-脯氨酸（150 mg，1.3 mmol）和t- BuOK（621 mg，6.46 mmol）在DMSO（15 mL）中的混合物攪拌過夜。隨後用EtOAc（100 mL）稀釋混合物，用水（30 mL×2）、飽和食鹽水（30 mL×1）洗滌，通過Na2 SO4 進行乾燥，過濾混合物並濃縮濾液，殘餘物通過正相矽膠柱層析純化，以PE/EtOAc=2/1（v/v）洗脫，得到無色油狀物2-((1-(4-甲氧基苄基)-4-((四氫-2H -吡喃-4-基)氨基)-1H -吡唑並[4,3-c ]吡啶-3-基)(甲基)氨基)乙醇（760 mg，57%）。m/z (ESI)+ : 412 [M+H]+ 。步驟 2 Synthesis of 2 - ((1-(4 -methoxybenzyl )-4 - (( tetrahydro- 2 H - pyran- 4 -yl ) amino )-1 H - pyrazolo [4,3- c ] Pyridin- 3 -yl )( methyl ) amino ) ethanol ( 40 ) 3-iodo-1-(4-methoxybenzyl)-N-(tetrahydro- 2H -pyran- at 100 °C 4-yl)-1 H -pyrazolo[4,3- c ]pyridin-4-amine (1.5 g, 3.23 mmol), 2-(methylamino)ethanol (1.98 g, 32.3 mmol), iodide A mixture of copper (123 mg, 0.65 mmol), L-valine (150 mg, 1.3 mmol) and t- BuOK (621 mg, 6.46 mmol) in DMSO (15 mL) was stirred overnight. Mixture was then diluted with water (30 mL × 2) with EtOAc (100 mL), saturated brine (30 mL × 1), dried over Na 2 SO 4 dried, the mixture was filtered and the filtrate concentrated, the residue was purified by normal phase silica gel column purified by chromatography to PE / EtOAc = 2/1 ( v / v) to give a colorless oil 2 - ((1- (4-methoxybenzyl) -4 - ((tetrahydro -2 H -pyran-4-yl)amino)-1 H -pyrazolo[4,3- c ]pyridin-3-yl)(methyl)amino)ethanol (760 mg, 57%). m/z (ESI) + : 412 [M+H] + . Step 2
合成 2 -((1-(4- 甲氧基苄基 )-4 -(( 四氫 -2H - 吡喃 -4- 基 ) 氨基 )-1H - 吡唑並 [4,3-c ] 吡啶 -3- 基 )( 甲基 ) 氨基 ) 甲磺酸乙酯（ 41 ） 在0℃下N2 保護，將甲磺醯氯（429 mg，3.7 mmol）逐滴地加入至2-((1-(4-甲氧基苄基)-4-((四氫-2H -吡喃-4-基)氨基)-1H -吡唑並[4,3-c ]吡啶-3-基)(甲基)氨基)乙醇（760 mg，1.85 mmol）和Et3 N（560 mg，5.55 mmol）的THF（76 mL）溶液中，隨後在室溫下將混合物攪拌0.5小時。用水（10 mL）淬滅反應並用EtOAc（20 mL×2）萃取，用飽和食鹽水（20 mL×1）洗滌合併的有機相，並通過Na2 SO4 進行乾燥，過濾有機相並濃縮濾液，殘餘物通過正相矽膠柱層析純化，PE/EtOAc=3:1（v/v）洗脫，得到無色油狀物2-((1-(4-甲氧基苄基)-4-((四氫-2H -吡喃-4-基)氨基)-1H -吡唑並[4,3-c ]吡啶-3-基)(甲基)氨基)甲磺酸乙酯（489 mg，54%）。m/z (ESI)+ : 490 [M+H]+ 。步驟 3 Synthesis of 2 - ((1-(4 -methoxybenzyl )-4 - (( tetrahydro- 2 H - pyran- 4 -yl ) amino )-1 H - pyrazolo [4,3- c ] Ethyl pyridin- 3 -yl )( methyl ) amino ) methanesulfonate ( 41 ) was N 2 protected at 0 ° C, and methyl sulfonium chloride (429 mg, 3.7 mmol) was added dropwise to 2-((1) -(4-methoxybenzyl)-4-((tetrahydro- 2H -pyran-4-yl)amino)-1 H -pyrazolo[4,3- c ]pyridin-3-yl) (methyl) amino) ethanol (760 mg, 1.85 mmol) and Et 3 N (560 mg, 5.55 mmol) in THF (76 mL) solution and then the mixture was stirred at room temperature for 0.5 hours. Washed with water (10 mL) quenched the reaction and extracted with EtOAc (20 mL × 2), washed with saturated brine (20 mL × 1) an organic phase, and through Na 2 SO 4 dried, filtered, the organic phase and the filtrate was concentrated The residue was purified by EtOAc EtOAc EtOAc (EtOAc) (tetrahydro-2 H -pyran-4-yl)amino)-1 H -pyrazolo[4,3- c ]pyridin-3-yl)(methyl)amino)methanesulfonate (489 mg , 54%). m/z (ESI) + : 490 [M+H] + . Step 3
合成 2-(4- 甲氧基苄基 )-9- 甲基 -6-( 四氫 -2H - 吡喃 -4- 基 )-6,7,8,9- 四氫 -2H -1,2,5,6,9- 五氮雜苯並 [cd ] 薁（ 42 ） 將t- BuOK（312 mg，2.79 mmol）加入至2-((1-(4-甲氧基苄基)-4-((四氫-2H -吡喃-4-基)氨基)-1H -吡唑並[4,3-c ]吡啶-3-基)(甲基)氨基)甲磺酸乙酯（455 mg，0.93 mmol）的無水THF（50 mL）溶液中。在N2 下，將反應物加熱至60℃反應1小時。隨後用水（10 mL）淬滅反應並用EtOAc（100 mL）萃取，用飽和食鹽水（30 mL）洗滌有機相，並且通過Na2 SO4 進行乾燥，過濾有機相，減壓濃縮濾液，殘餘物通過正相矽膠柱層析純化，PE/EtOAc=1/1（v/v）洗脫，得到油狀物2-(4-甲氧基苄基)-9-甲基-6-(四氫-2H -吡喃-4-基)-6,7,8,9-四氫-2H -1,2,5,6,9-五氮雜苯並[cd ]薁（167 mg，46%）。1 H-NMR (400 MHz, CDCl3 )δ ppm 1.57-1.66 (m, 2H), 1.79 (m, 2H), 3.06 (s, 3H), 3.37 (m, 2H), 3.60 (m, 4H), 3.77 (s, 3H), 4.04 (m, 2H), 5.24 (m, 3H), 6.37 (d,J = 6.0 Hz, 1H), 6.82 (d,J = 8.0 Hz, 2H), 7.16 (d,J = 8.0 Hz, 2H), 7.78 (d,J = 6.0 Hz, 1H); m/z (ESI)+ : 394 [M+H]+ 。步驟 4 Synthesis of 2-(4 -methoxybenzyl )-9- methyl -6-( tetrahydro- 2 H - pyran- 4 -yl )-6,7,8,9 -tetrahydro- 2 H - 1 , 2,5,6,9 - pentazabenzo [ cd ] 薁 ( 42 ) t- BuOK (312 mg, 2.79 mmol) was added to 2-((1-(4-methoxybenzyl)- 4-((tetrahydro-2 H -pyran-4-yl)amino)-1 H -pyrazolo[4,3- c ]pyridin-3-yl)(methyl)amino)methanesulfonate (455 mg, 0.93 mmol) in dry THF (50 mL). The reaction was heated to 60 ° C for 1 hour under N 2 . Then washed with water (10 mL) quenched the reaction and extracted with EtOAc (100 mL), washed with saturated brine (30 mL) The organic phase was washed, and by Na 2 SO 4 dried, filtered and the organic phase was concentrated under reduced pressure and the filtrate, the residue was purified by Purification by column chromatography on silica gel eluting with PE/EtOAc = 1/1 (v/v) afforded 2-(4-methoxybenzyl)-9-methyl-6-(tetrahydro- 2 H -pyran-4-yl)-6,7,8,9-tetrahydro-2 H -1,2,5,6,9-pentazabenzo[ cd ]薁 (167 mg, 46% ). 1 H-NMR (400 MHz, CDCl 3 ) δ ppm 1.57-1.66 (m, 2H), 1.79 (m, 2H), 3.06 (s, 3H), 3.37 (m, 2H), 3.60 (m, 4H), 3.77 (s, 3H), 4.04 (m, 2H), 5.24 (m, 3H), 6.37 (d, J = 6.0 Hz, 1H), 6.82 (d, J = 8.0 Hz, 2H), 7.16 (d, J = 8.0 Hz, 2H), 7.78 (d, J = 6.0 Hz, 1H); m/z (ESI) + : 394 [M+H] + . Step 4
合成 9- 甲基 -6-( 四氫 -2H - 吡喃 -4- 基 )-6,7,8,9- 四氫 -2H -1,2,5,6,9- 五氮雜苯並 [cd ] 薁（實施例 93 ） 在N2 下，將2-(4-甲氧基苄基)-9-甲基-6-(四氫-2H -吡喃-4-基)-6,7,8,9-四氫-2H -1,2,5,6,9-五氮雜苯並[cd ]薁（150 mg，0.38 mmol）的TFA（7.5 mL）溶液加熱至70℃反應8小時，後減壓濃縮除去TFA，將殘餘物溶解於EtOAc（150 mL）中，用NaHCO3 水溶液（20 mL×2）、飽和食鹽水（20 mL×1）洗滌，通過Na2 SO4 進行乾燥，過濾後濃縮濾液，殘餘物通過製備TLC（DCM/MeOH=11/1，v/v）純化，得到白色固體9-甲基-6-(四氫-2H -吡喃-4-基)-6,7,8,9-四氫-2H -1,2,5,6,9-五氮雜苯並[cd ]薁（75 mg，72%）。1 H-NMR (400 MHz, DMSO-d6 )δ ppm 1.71 (m, 2H), 1.87 (m, 2H), 3.02(s, 3H), 3.44 (m, 4H), 3.78(m, 2H), 3.98 (m, 2H), 4.33(m, 1H), 6.84(s, 1H), 7.55(s, 1H), 12.35 (br s, 1H), 12.98 (br s, 1H);19 F-NMR (376 MHz, DMSO-d6 )δ ppm -74.41; m/z (ESI)+ : 274 [M+H]+ 。實施例 94 Synthesis of 9 -methyl -6-( tetrahydro- 2 H - pyran- 4 -yl )-6,7,8,9 -tetrahydro- 2 H - 1,2,5,6,9 -pentaza Benzo [ cd ] oxime (Example 93 ) 2-(4-methoxybenzyl)-9-methyl-6-(tetrahydro-2 H -pyran-4-yl) under N 2 a solution of -6,7,8,9-tetrahydro-2 H -1,2,5,6,9-pentazabenzo[ cd ] oxime (150 mg, 0.38 mmol) in TFA (7.5 mL) the reaction 70 ℃ 8 hours. after concentrated under reduced pressure to remove TFA, the residue was dissolved in (150 mL) in EtOAc, washed with aqueous NaHCO 3 (20 mL × 2), saturated brine (20 mL × 1), Na 2 by SO 4 dried, filtered, and the filtrate was concentrated, the residue was purified by preparative TLC (DCM / MeOH = 11/ 1, v / v), to give a white solid 9-methyl-6- (tetrahydro -2 H - pyran - 4-yl)-6,7,8,9-tetrahydro- 2H -1,2,5,6,9-pentazabenzo[ cd ]indole (75 mg, 72%). 1 H-NMR (400 MHz, DMSO- d 6 ) δ ppm 1.71 (m, 2H), 1.87 (m, 2H), 3.02 (s, 3H), 3.44 (m, 4H), 3.78 (m, 2H), 3.98 (m, 2H), 4.33 (m, 1H), 6.84 (s, 1H), 7.55 (s, 1H), 12.35 (br s, 1H), 12.98 (br s, 1H); 19 F-NMR (376 MHz, DMSO- d 6 ) δ ppm -74.41; m/z (ESI) + : 274 [M+H] + . Example 94
在步驟1中使用2-(乙基氨基)乙醇代替2-(甲基氨基)乙醇，類似於實施例93
地製備實施例94
。
使用以下方法，測定實施例的LRRK2抑制和對其它激酶如JAK2的選擇性。材料和方法 材料 The LRRK2 inhibition of the examples and the selectivity to other kinases such as JAK2 were determined using the following methods. Materials and method materials
在Adapta™激酶測定法（LRRK2_IC50 測定）中：激酶反應緩衝液含有5×激酶緩衝液S（Life Technologies，PV5213）和2 mM DTT（Life Technologies，P2325）。激酶LRRK2 G2019S蛋白（PV4881）和ERM（LRRKtide，PV5093）源自Life Technologies。Adapta™通用激酶測定試劑盒（Life Technologies，PV5099）包含以下組分：Adapta™Eu-抗-ADP抗體（PV5097；4μg）；10 μM AlexaFluor®647 ADP示蹤劑（PV5098；200 pmol）；TR-FRET稀釋緩衝液（PV3574；100ml）；激酶淬滅緩衝液（P2825；1ml）；10mM ATP（PV3227；500μl）；和10mM ADP（PV5096；500μl）。LRRK2-IN-1（1234480-84-2，HY-10875）源自MedChem Express。In Adapta ™ kinase assay (LRRK2_IC 50 assay): Kinase reaction buffer containing 5 × kinase buffer S (Life Technologies, PV5213) and 2 mM DTT (Life Technologies, P2325 ). The kinases LRRK2 G2019S protein (PV4881) and ERM (LRRKtide, PV5093) were derived from Life Technologies. The AdaptaTM Universal Kinase Assay Kit (Life Technologies, PV5099) contains the following components: AdaptaTM Eu-anti-ADP antibody (PV5097; 4 μg); 10 μM AlexaFluor® 647 ADP tracer (PV5098; 200 pmol); TR- FRET dilution buffer (PV3574; 100 ml); kinase quenching buffer (P2825; 1 ml); 10 mM ATP (PV3227; 500 μl); and 10 mM ADP (PV5096; 500 μl). LRRK2-IN-1 (1234480-84-2, HY-10875) was derived from MedChem Express.
在LANCE® 激酶測定法（JAK2_IC50 選擇性測定）中：JAK2（Invitrogen，目錄號PV4288），ATP（Sigma，目錄號A7699-1 g），DMSO（Sigma，目錄號D2650），DTT（Sigma，目錄號43815），LANCE Ultra ULightTM -JAK-1肽（Perkin Elmer，目錄號TRF0121），LANCE Eu-W1024抗磷酸酪氨酸（PT66）（Perkin Elmer，目錄號AD0069），LANCETM檢測緩衝液（Perkin Elmer，目錄號CR97-100），Tofacitinib（PharmaBlock Sciences（Nanjing），Inc,目錄號PBN2011586-01）；設備：Envision（Perkin Elmer），Bravo（Agilent）；消耗品：384孔中間板（Greiner，目錄號781280），384孔測定板（Perkin Elmer，目錄號6007299），In the LANCE ® assay kinase (JAK2_IC 50 selectivity assay): JAK2 (Invitrogen, Cat # PV4288), ATP (Sigma, catalog number A7699-1 g), DMSO (Sigma, catalog No. D2650), DTT (Sigma, catalog No. 43815), LANCE Ultra ULight TM -JAK -1 peptide (Perkin Elmer, Catalog No. TRF0121), LANCE Eu-W1024 anti-phosphotyrosine (the PT66) (Perkin Elmer, Cat. No. AD0069), LANCETM assay buffer (Perkin Elmer , catalog number CR97-100), Tofacitinib (PharmaBlock Sciences (Nanjing), Inc, catalog number PBN2011586-01); Equipment: Envision (Perkin Elmer), Bravo (Agilent); Consumables: 384-well intermediate plate (Greiner, catalog number 781280), 384-well assay plate (Perkin Elmer, catalog number 6007299),
在果蠅模型中：抗LRRK2（phospho S935）抗體[UDD2 10(12)]（ab133450）源自Abcam。ddc-GAL4源自蘇州大學醫學系。Adapta™ 激酶試驗方法 In the Drosophila model: anti-LRRK2 (phospho S935) antibody [UDD2 10(12)] (ab133450) was derived from Abcam. ddc-GAL4 is from the Department of Medicine of Suzhou University. AdaptaTM Kinase Test Method
Adapta®通用激酶測定法是一種用於檢測ADP的均勻的基於螢光的免疫測定法。與ATP消耗測定法相反，Adapta®測定法對ADP形成非常敏感，大多數信號能夠在初始10-20%的ATP轉化為ADP時改變發生。這使得Adapta®通用激酶測定法非常適合用於低活性激酶。The Adapta® Universal Kinase Assay is a homogeneous fluorescence-based immunoassay for the detection of ADP. In contrast to the ATP depletion assay, the Adapta® assay is very sensitive to ADP formation, and most signals can change when the initial 10-20% ATP is converted to ADP. This makes the Adapta® Universal Kinase Assay ideal for use in low activity kinases.
在室溫（〜21℃）下進行所有測定，並且在所用條件下該測定與時間和酶濃度呈線性關係。由5×激酶緩衝液S儲存液（以上列出的）製備1×激酶反應緩衝液溶液，通過向8ml H2 O中加入2ml 5×儲存液來製備10 ml的1×激酶反應緩衝液。將20 μl的1 M DTT加入至此1×激酶反應緩衝液中。All assays were performed at room temperature (~21 °C) and the assay was linear with time and enzyme concentration under the conditions used. A 5 × kinase buffer storage solution S (listed above) Preparation of 1 × kinase reaction buffer solution, prepared by adding 5 × 2ml stock solution to 8ml H 2 O in 10 ml of 1 × kinase reaction buffer. 20 μl of 1 M DTT was added to this 1× kinase reaction buffer.
在低容量384孔板中以10μl體積進行激酶反應。通常，使用Greiner板（目錄＃3674＃，低容量，白壁，784075）。未經測試的其它未經處理的測定板也可能是合適的。這種測定中的底物濃度為100 μM，且1×激酶反應緩衝液由50 mM Tris-HCl pH8.5、10mM MgCl2 、1 mM EGTA、0.01%Brij-35和2mM DTT，以及任何其它的可能是特定激酶所必需的添加劑。允許激酶反應在室溫下進行1小時，隨後添加5 μl的TR-FRET稀釋緩衝液中的激酶淬滅緩衝液（EDTA；30mM）、Eu標記的抗體（6nM）和示蹤劑（18.9nM）的製劑。測定孔中的抗體的最終濃度為2nM，示蹤劑為6.3nM且EDTA為10mM。允許板在室溫下平衡至少30分鐘，隨後在配置用於AdaptaTM TR-FRET的平板讀取儀上讀取。The kinase reaction was carried out in a low volume 384 well plate in a volume of 10 μl. Usually, use the Greiner board (catalog #3674#, low capacity, white wall, 784075). Other untreated assay plates that have not been tested may also be suitable. The substrate concentration in this assay was 100 μM and the 1× kinase reaction buffer consisted of 50 mM Tris-HCl pH 8.5, 10 mM MgCl 2 , 1 mM EGTA, 0.01% Brij-35 and 2 mM DTT, and any other May be an additive necessary for a particular kinase. The kinase reaction was allowed to proceed for 1 hour at room temperature, followed by the addition of 5 μl of kinase quenching buffer (EDTA; 30 mM), Eu-labeled antibody (6 nM) and tracer (18.9 nM) in TR-FRET dilution buffer. Preparation. The final concentration of antibody in the assay well was 2 nM, the tracer was 6.3 nM and the EDTA was 10 mM. Allow plates were equilibrated for at least 30 minutes at room temperature, and then read on a plate reader configured to Adapta TM TR-FRET.
使用BMG LABTECH PHERAstar平板讀取儀，使用用於Adapta™的適當濾波器和儀器設置產生本文檔中提供的資料。在孔中以1%DMSO（最終）篩選測試化合物。對於8點滴定，從起始濃度進行5次連續稀釋。LANCE® 激酶選擇性試驗方法 Use the BMG LABTECH PHERAstar Tablet Reader to generate the information provided in this document using the appropriate filters and instrument settings for AdaptaTM. Test compounds were screened in wells in 1% DMSO (final). For the 8-point titration, 5 serial dilutions were performed from the starting concentration. LANCE ® kinase selectivity test method
該測定法涉及兩個步驟，酶促步驟和用HTRF試劑進行的檢測步驟。步驟1：在酶促步驟期間，將底物生物素與感興趣的激酶一起孵育。加入ATP，開始反應。步驟2：檢測試劑捕獲磷酸化底物，所得TR-FRET與磷酸化水準成比例。The assay involves two steps, an enzymatic step and a detection step with an HTRF reagent. Step 1: The substrate biotin is incubated with the kinase of interest during the enzymatic step. Add ATP and start the reaction. Step 2: The detection reagent captures the phosphorylated substrate and the resulting TR-FRET is proportional to the phosphorylation level.
化合物製備：將測試化合物溶解於30mM DMSO溶液中，並在室溫下將此溶液放入氮氣罩中長期儲存；以3倍係數，總共11種濃度，稀釋DMSO中的30mM化合物；吸出1μl化合物，隨後用激酶緩衝液將該化合物稀釋25倍。混合均勻，室溫下平衡30分鐘。Compound preparation: The test compound was dissolved in a 30 mM DMSO solution, and the solution was placed in a nitrogen blanket for a long period of time at room temperature; 30 mM compound in DMSO was diluted at a total factor of 11 with a factor of 3; 1 μl of the compound was aspirated, This compound was then diluted 25-fold with kinase buffer. Mix well and equilibrate for 30 minutes at room temperature.
激酶反應：通過Agilent Bravo，將2.5μl激酶緩衝液（4x）中的化合物轉移到測定板中。旋轉板；通過使用Eppendorf電子多通道移液器，將5μl酶混合物轉移到測定板中。旋轉板；在室溫（23℃）下孵育測定板20分鐘；通過使用Multidrop，將2.5 μl的含有ATP的激酶緩衝液加入至測定板中。旋轉並密封板；在室溫（23℃）下孵育測定板90分鐘。Kinase reaction: Compounds in 2.5 [mu]l Kinase Buffer (4x) were transferred to assay plates by Agilent Bravo. The plate was rotated; 5 μl of the enzyme mixture was transferred to the assay plate by using an Eppendorf electronic multichannel pipette. The plate was rotated; the assay plate was incubated for 20 minutes at room temperature (23 ° C); 2.5 μl of ATP-containing kinase buffer was added to the assay plate by using Multidrop. The plate was rotated and sealed; the assay plate was incubated for 90 minutes at room temperature (23 ° C).
停止反應：使用Eppendorf電子多通道移液器，將10μl檢測試劑（2nM LANCE Eu-W1024抗磷酸酪氨酸）轉移至測定板中。旋轉並密封板；在室溫（23℃）下孵育測定板60分鐘。The reaction was stopped: 10 μl of detection reagent (2 nM LANCE Eu-W1024 anti-phosphotyrosine) was transferred to the assay plate using an Eppendorf electronic multichannel pipette. The plate was rotated and sealed; the assay plate was incubated for 60 minutes at room temperature (23 ° C).
檢測和讀取：激發波長為340 nm，初次發射波長為615 nm，二次發射波長為665 nm（分別用於Cryptate和Ulight）。用EnVision讀板，得到兩個波長的讀數；計算665 nm/615 nm的比率。Detection and reading: The excitation wavelength is 340 nm, the initial emission wavelength is 615 nm, and the secondary emission wavelength is 665 nm (for Cryptate and Ulight, respectively). Use EnVision to read the plate and get two wavelength readings; calculate the ratio of 665 nm / 615 nm.
使用XLfit（IDBS Inc.）軟體，獲得IC50 估計值。果蠅模型篩選方法 IC 50 estimates were obtained using XLfit (IDBS Inc.) software. Drosophila model screening method
果蠅模型用於在體內評價實例。由Andrea Brand和Norbert Perrimon於1993年開發的GAL4/UAS系統[45]用於產生表達多巴胺（DA）神經元中的LRRK2-G2019S的轉基因果蠅。該系統有兩個部分：編碼酵母轉錄啟動蛋白GAL4的GAL4基因和GAL4特異性結合啟動基因轉錄的增強子UAS（上游啟動序列）。該系統利用了與UAS特異性結合的酵母GAL4轉錄因數。將UAS-野生型-LRRK2和UAS-G2019S-LRRK2轉基因與多巴脫羧酶（ddc ）-GAL4驅動子[46]組合，以表達DA神經元中的LRRK2-G2019S。10 μM的GW5074用於陽性對照。陰性對照組為DMSO對照（所有化合物以1:1000稀釋溶解於DMSO中）。在12小時光照12小時黑暗迴圈中將果蠅保持在25℃。GW5074用作為陽性對照[47]。 存活率The Drosophila model is used to evaluate examples in vivo. The GAL4/UAS system [45] developed by Andrea Brand and Norbert Perrimon in 1993 was used to generate transgenic flies expressing LRRK2-G2019S in dopamine (DA) neurons. The system has two components: the GAL4 gene encoding the yeast transcriptional promoter protein GAL4 and the enhancer UAS (upstream promoter sequence) that specifically binds to the promoter of GAL4. This system utilizes the yeast GAL4 transcription factor that specifically binds to UAS. The UAS-wild-LRRK2 and UAS-G2019S-LRRK2 transgenes were combined with the dopa decarboxylase ( ddc )-GAL4 driver [46] to express LRRK2-G2019S in DA neurons. 10 μM of GW5074 was used for the positive control. The negative control group was a DMSO control (all compounds were dissolved in DMSO at a 1:1000 dilution). The fruit flies were maintained at 25 °C in a 12 hour light 12 hour dark loop. GW5074 was used as a positive control [47]. Survival rate
收集二十隻最新封閉的雌性果蠅，並將其放入食物小瓶中。每隔一天將果蠅轉移到新鮮的食物小瓶中，此時記錄死亡。Twenty of the newly closed female fruit flies were collected and placed in food vials. The fruit flies were transferred to fresh food vials every other day and death was recorded.
基於果蠅的生存曲線，50%存活時間參數表示存活一半果蠅的時間，並且將此參數用於比較不同組之間的存活率。基於每組的4個獨立實驗，計算平均50%存活時間和標準誤差。通過GraphPad PRISM® 6.0軟體分析資料。 攀爬試驗（Climbing Assay）Based on the Drosophila survival curve, the 50% survival time parameter indicates the time to survive half of the fruit fly, and this parameter was used to compare survival between different groups. On average, 50% survival time and standard error were calculated based on 4 independent experiments per group. Analyze data with GraphPad PRISM ® 6.0 software. Climbing Assay
負趨地性測定用於分析果蠅的運動能力。讓每小瓶的二十隻果蠅（每組四隻小瓶）每週進行一次攀爬試驗。Negative geodetic assays were used to analyze the motor ability of fruit flies. Twenty fruit flies per bottle (four vials per group) were tested weekly for climbing.
將待測試的果蠅轉移至垂直塑膠管（15 cm高，1.5 cm的直徑）中。在室溫下靜置30分鐘後，將果蠅輕輕敲打至管底，計數10秒內可以爬至或高於測試線的果蠅數目，並且計算百分比。Transfer the fruit flies to be tested to a vertical plastic tube (15 cm high, 1.5 cm diameter). After standing at room temperature for 30 minutes, the fruit fly was gently tapped to the bottom of the tube, and the number of fruit flies that could climb to or above the test line within 10 seconds was counted, and the percentage was calculated.
基於每個小瓶的三次獨立實驗分析攀爬能力，並通過GraphPad PRISM® 6.0軟體分析資料。 在第6周進行的激酶試驗Climbing capabilities were analyzed based on three independent experiments per vial and analyzed by GraphPad PRISM ® 6.0 software. Kinase assay performed at week 6
在冰上將成體果蠅頭均質化，在具有ATP和DTT的激酶反應緩衝液中進行腦裂解物的激酶反應。The adult fruit fly head was homogenized on ice, and the kinase reaction of brain lysate was carried out in a kinase reaction buffer with ATP and DTT.
隨後，通過12%SDS-PAGE凝膠，使裂解物進行電泳，並轉移到PVDF膜（Millipore）。在室溫下，在具有5%脫脂乳的TBST中封閉該膜1小時，隨後在4℃下在抗LRRK2 pSer935抗體（Abcam，ab133450）和抗Flag抗體中孵育過夜。Subsequently, the lysate was subjected to electrophoresis through a 12% SDS-PAGE gel and transferred to a PVDF membrane (Millipore). The membrane was blocked in TBST with 5% skim milk for 1 hour at room temperature, followed by incubation in anti-LRRK2 pSer935 antibody (Abeam, ab133450) and anti-Flag antibody overnight at 4 °C.
用HRP綴合的二抗和ECL檢測試劑檢測蛋白質。通過Image J軟體分析免疫印跡的光密度，並計算磷酸化LRRK2蛋白相比於Flag蛋白的比率，並通過GraphPad PRISM® 6.0軟體分析資料。激酶選擇性試驗方法 1. MKK1 試驗 Proteins were detected using HRP-conjugated secondary antibodies and ECL detection reagents. Immunoblot analysis of the optical density by Image J software, and calculates the LRRK2 protein phosphorylation compared to protein ratio Flag, and analyze the data by GraphPad PRISM ® 6.0 software. Kinase selectivity test method 1. MKK1 test
這是一種兩步測定法，其中在25.5 μl的含有25 mM Tris、0.1 mM EGTA、0.01%Brij35、10 mM乙酸鎂和0.005 mM ATP中，無活性的MAPK（0.06 mg/ml）被MKK1（稀釋於25mM Tris、0.1 mM EGTA、0.1%b-巰基乙醇、0.01%Brij35、1 mg/ml BSA中）啟動。在室溫下孵育30分鐘後，從第一次反應移液5μl至20μl的含有（終濃度）25mM Tris pH 7.5、0.1 mM EGTA、0.1 mM Na3VO4、0.66 mg/ml髓鞘鹼性蛋白（MBP）、10 mM乙酸鎂和0.05 mM[33P-g-ATP]（500-1000 cpm/pmole）的第二反應混合物中，並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集至P81 Unifilter板上。2. MAPK2 /ERK2 試驗 This is a two-step assay in which inactive MAPK (0.06 mg/ml) is diluted by MKK1 in 25.5 μl of 25 mM Tris, 0.1 mM EGTA, 0.01% Brij35, 10 mM magnesium acetate and 0.005 mM ATP. Initiation in 25 mM Tris, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 0.01% Brij 35, 1 mg/ml BSA). After incubation for 30 minutes at room temperature, 5 μl to 20 μl of the first reaction was dispensed (final concentration) 25 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM Na3VO4, 0.66 mg/ml myelin basic protein (MBP) , 10 mM magnesium acetate and 0.05 mM [33P-g-ATP] (500-1000 cpm/pmole) in the second reaction mixture and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid, which was then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 2. MAPK2 / ERK2 test
在25.5 µl終體積的25 mM Tris pH 7.5、0.1 mM EGTA、0.33 mg/ml MBP、10mM乙酸鎂和0.05mM[33P-g-ATP]（500-1000 cpm/pmole）中，測定針對於MBP的MAPK/ERK2（5–20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1 mM Na3VO4、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集至P81 Unifilter板中。3. JNK1a1 /SAPK1c 試驗 Determination of MBP in a final volume of 25.5 μl of 25 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate and 0.05 mM [33P-g-ATP] (500-1000 cpm/pmole) MAPK/ERK2 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM Na3VO4, 0.1% b-mercaptoethanol, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid, which was then collected into a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 3. JNK1a1 / SAPK1c test
在25.5 µl終體積的50 mM Tris pH 7.5、0.1 mM EGTA、0.1%b-巰基乙醇、ATF2（3μM）、10mM乙酸鎂和0.02mM[33P-g-ATP]（500-1000cpm/pmole）中，測定針對於ATF2（啟動轉錄因數）的JNK1a1/SAPK1c（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、1 mg/ml BSA、0.1%b-巰基乙醇中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。4. SAPK 2a /p38 試驗 In a final volume of 25.5 μl of 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, ATF2 (3 μM), 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (500-1000 cpm/pmole), Determination of JNK1a1/SAPK1c (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA, 0.1% b-mercaptoethanol) against ATF2 (starting transcription factor) and at room temperature Incubate for 30 minutes. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 4. SAPK 2a / p38 test
在含有25 mM Tris pH 7.5、0.1 mM EGTA、0.33 mg/ml MBP、10mM乙酸鎂和0.05 mM[33P-g-ATP]（500-1000cpm/pmole）的25.5 µl終體積中，測定針對於MBP的SAPK 2a/p38（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1 mM Na3VO4、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。5. SAPK 2b/p38ß2 試驗 Determination of MBP in a final volume of 25.5 μl containing 25 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate and 0.05 mM [33P-g-ATP] (500-1000 cpm/pmole) SAPK 2a/p38 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM Na3VO4, 0.1% b-mercaptoethanol, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 5. SAPK 2b/p38ß2 test
在含有25 mM Tris pH 7.5、0.1 mM EGTA、0.33 mg/ml MBP、10mM乙酸鎂和0.02 mM[33P-g-ATP]（500-1000cpm/pmole）的25.5 µl終體積中，測定針對於MBP的SAPK 2b/p38ß2（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1 mM Na3VO4、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。6. SAPK 3/p38 g 試驗 Determination of MBP for MBP in a final volume of 25.5 μl containing 25 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (500-1000 cpm/pmole) SAPK 2b/p38ß2 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM Na3VO4, 0.1% b-mercaptoethanol, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 6. SAPK 3/p38 g test
在含有25 mM Tris pH 7.5、0.1 mM EGTA、0.33 mg/ml MBP、10mM乙酸鎂和0.005 mM[33P-g-ATP]（500-1000cpm/pmole）的25.5 µl終體積中，測定針對於MBP的SAPK 3/p38 g（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1 mM Na3VO4、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。7. SAPK 4/p38∂ 試驗 Determination of MBP in a final volume of 25.5 μl containing 25 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (500-1000 cpm/pmole) SAPK 3/p38 g (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM Na3VO4, 0.1% b-mercaptoethanol, 1 mg/ml BSA) and incubated for 30 minutes at room temperature . The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 7. SAPK 4/p38∂ test
在含有25 mM Tris pH 7.5、0.1 mM EGTA、0.33 mg/ml MBP、10mM乙酸鎂和0.005 mM[33P-g-ATP]（500-1000cpm/pmole）的25.5 µl終體積中，測定針對於MBP的SAPK 4/p38d（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1 mM Na3VO4、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。8. MAPKAP-K1a 試驗 Determination of MBP in a final volume of 25.5 μl containing 25 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (500-1000 cpm/pmole) SAPK 4/p38d (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM Na3VO4, 0.1% b-mercaptoethanol, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 8. MAPKAP-K1a test
在含有50 mM Na-b-甘油磷酸鹽 pH 7.5、0.5 mM EDTA、30 µM底物肽、10 mM乙酸鎂和0.05 mM[33P-g-ATP](50-1000 cpm/pmole)的25.5 µl終體積中，測定針對於KKLNRTLSVA的MAPKAP-K1a（5-20 mU，稀釋於20 mM MOPS pH 7.5、1 mM EDTA、0.01% Brij35、5%甘油、0.1% b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育40分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。9. MAPKAP-K2 試驗 At 25.5 μl final containing 50 mM Na-b-glycerophosphate pH 7.5, 0.5 mM EDTA, 30 μM substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) In the volume, MAPKAP-K1a (5-20 mU for KKLNRTLSVA, diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01% Brij35, 5% glycerol, 0.1% b-mercaptoethanol, 1 mg/ml BSA) ) and incubate for 40 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 9. MAPKAP-K2 test
在含有50 mM Na-b-甘油磷酸鹽 pH 7.5、0.5 mM EDTA、30 µM底物肽、10 mM乙酸鎂和0.02 mM[33P-g-ATP](50-1000 cpm/pmole)的25.5 µl終體積中，測定針對於KKLNRTLSVA的MAPKAP-K2（5-20 mU，稀釋於20 mM MOPS pH 7.5、1 mM EDTA、0.01% Brij35、5%甘油、0.1% b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。10. MSK1 試驗 At 25.5 μl final containing 50 mM Na-b-glycerophosphate pH 7.5, 0.5 mM EDTA, 30 μM substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) In the volume, MAPKAP-K2 (5-20 mU for KKLNRTLSVA, diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01% Brij35, 5% glycerol, 0.1% b-mercaptoethanol, 1 mg/ml BSA) ) and incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 10. MSK1 test
在含有8 mM MOPS pH7.0、0.2 mM EDTA、30 µM底物肽、10 mM乙酸鎂和0.02 mM[33P-g-ATP](50-1000 cpm/pmole)的25.5 µl終體積中，測定針對於經修飾的Crosstide肽GRPRTSSFAEGKK的MSK1（5-20 mU，稀釋於20 mM MOPS pH 7.5、1 mM EDTA、0.01% Brij35、0.1% b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。11. PRAK 試驗 In a final volume of 25.5 μl containing 8 mM MOPS pH 7.0, 0.2 mM EDTA, 30 μM substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole), MSK1 (5-20 mU, diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01% Brij35, 0.1% b-mercaptoethanol, 1 mg/ml BSA) of the modified Crosstide peptide GRPRTSSFAEGKK, and at room temperature Incubate for 30 minutes. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 11. PRAK test
在含有50 mM b-甘油磷酸鈉pH 7.5、0.1 mM EGTA、30μM底物肽、10mM乙酸鎂和0.02mM[33P-g-ATP]（50-1000cpm/pmole）的25.5μl終體積中，測定針對於KKLRRTLSVA的PRAK（5-20 mU，稀釋於50 mM b-甘油磷酸鈉pH 7.5、0.1 mM EGTA、0.1% b-巰基乙醇、1 mg/ml BSA），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。12. PKA 試驗 In a final volume of 25.5 μl containing 50 mM b-glycerophosphate pH 7.5, 0.1 mM EGTA, 30 μM substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole), PRAK in KKLRRTLSVA (5-20 mU, diluted in 50 mM b-glycerophosphate pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 12. PKA test
在含有8 mM MOPS pH 7.5、0.2 mM EDTA、30 µM底物肽、10 mM乙酸鎂和0.005 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5μl終體積中，測定針對於肯普肽（LRRASLG）的PKA（5-20 mU，稀釋於20 mM MOPS pH 7.5、1 mM EDTA、0.01% Brij35、0.1% b-巰基乙醇、1 mg/ml BSA），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。13. PKCa 試驗 In a final volume of 25.5 μl containing 8 mM MOPS pH 7.5, 0.2 mM EDTA, 30 μM substrate peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole), the assay was for Kappa peptide (LRRASLG) PKA (5-20 mU diluted to 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01% Brij35, 0.1% b-mercaptoethanol, 1 mg/ml BSA) and incubated at room temperature 30 minutes. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 13. PKCa test
在磷脂醯絲氨酸（PtdSerine）和DAG（0.1 mg/ml和10μg/ml）以及0.1 mM CaCl2存在下，測定針對於組蛋白H1的PKCa（5-20 mU，稀釋於20mM Hepes pH 7.4，0.03%Triton X-100中）。該測定在25.5μl終體積中進行，該體積含有20mM Hepes pH 7.4、0.03%Triton X-100、0.1 mg/ml組蛋白H1、10mM乙酸鎂和0.02mM[33P-g-ATP]（50-1000cpm/pmole），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板中。Determination of PKCa against histone H1 in the presence of phospholipid serine (PtdSerine) and DAG (0.1 mg/ml and 10 μg/ml) and 0.1 mM CaCl2 (5-20 mU, diluted to 20 mM Hepes pH 7.4, 0.03% Triton) X-100). The assay was performed in a final volume of 25.5 μl containing 20 mM Hepes pH 7.4, 0.03% Triton X-100, 0.1 mg/ml histone H1, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm). /pmole) and incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid, which was then collected into a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.
製備PtdSer/DAG：-在MeOH/氯仿（1:2）中的PtdSer原料=10 mg/ml。乾燥所需量。重懸於適當體積的10mM Hepes pH 7.4中。渦旋並短暫地超音波。（2×10-15秒，間隔10-15秒）。在MeOH/氯仿（1:2）中的DAG原料=10 mg/ml。乾燥所需量。添加經超音波的PtdSer溶液。進行渦旋和超音波。14. PDK1 試驗 Preparation of PtdSer/DAG: - PtdSer starting material in MeOH/chloroform (1:2) = 10 mg/ml. Dry the required amount. Resuspend in an appropriate volume of 10 mM Hepes pH 7.4. Vortex and briefly supersonic. (2 × 10-15 seconds, interval 10-15 seconds). DAG starting material in MeOH/chloroform (1:2) = 10 mg/ml. Dry the required amount. Ultrasonic PtdSer solution was added. Perform vortex and ultrasonic waves. 14. PDK1 test
在含有50 mM Tris pH 7.5、0.05%%b-巰基乙醇、100 µM底物肽、10mM乙酸鎂和0.02 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5μl終體積中，測定針對於PDKtide（KTFCGTPEYLAPEVRREPRILSEEEQ-EMFRDFDYIADWC）的PDK1（5-20 mU，稀釋於50 mM Tris pH 7.5、0.05%%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。15. ΔPH-PKBa-S473D 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% b-mercaptoethanol, 100 μM substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole), PDK1 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.05%% b-mercaptoethanol, 1 mg/ml BSA) against PDKtide (KTFCGTPEYLAPEVRREPRILSEEEQ-EMFRDFDYIADWC) was assayed and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 15. ΔPH-PKBa-S473D Test
在含有50 mM Tris pH 7.5、0.05%b-巰基乙醇、30 µM底物肽、10 mM乙酸鎂和0.005 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於經修飾的Crosstide肽GRPRTSSFAEGKK的ΔPH-PKBa-S473D（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。16. SGK 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% b-mercaptoethanol, 30 μM substrate peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole), ΔPH-PKBa-S473D (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 1 mg/ml BSA) against the modified Crosstide peptide GRPRTSSFAEGKK was determined and Incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 16. SGK test
在含有8 mM MOPS pH 7.0、0.2 mM EDTA、30 µM底物肽、10 mM乙酸鎂和0.02 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於經修飾的Crosstide肽GRPRTSSFAEGKK的SGK（5-20 mU，稀釋於20 mM MOPS pH 7.5、1 mM EDTA、0.01% Brij35、5%甘油、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。17. S6K1/P70 S6K 試驗 In a final volume of 25.5 μl containing 8 mM MOPS pH 7.0, 0.2 mM EDTA, 30 μM substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole), the assay was for SGK (5-20 mU, modified in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01% Brij35, 5% glycerol, 0.1% b-mercaptoethanol, 1 mg/ml BSA) of the modified Crosstide peptide GRPRTSSFAEGKK, and Incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 17. S6K1/P70 S6K test
在含有8 mM MOPS pH 7.0、0.2 mM EDTA、0.1 mM底物肽、10 mM乙酸鎂和0.02 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（KKRNRTLTV）的S6K1/P70 S6K（5-20 mU，稀釋於20 mM MOPS pH 7.5、1 mM EDTA、0.01% Brij35、5%甘油、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。18. GSK3b 試驗 In a final volume of 25.5 μl containing 8 mM MOPS pH 7.0, 0.2 mM EDTA, 0.1 mM substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole), the assay was for Substrate peptide (KKRNRTLTV) S6K1/P70 S6K (5-20 mU, diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01% Brij35, 5% glycerol, 0.1% b-mercaptoethanol, 1 mg/ml BSA ) and incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 18. GSK3b test
在含有8 mM MOPS pH 7.0、0.2 mM EDTA、20 µM磷酸化GS2肽、10 mM乙酸鎂和0.005 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於磷酸化GS2肽（YRRAAVPPSPSLSRHSSPHQS(PO4)EDEEE）的GSK3b（5-20 mU，稀釋於20 mM MOPS pH 7.5、1 mM EDTA、0.01% Brij35、5%甘油、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。19. ROCK-II (ROKa) 試驗 In a final volume of 25.5 μl containing 8 mM MOPS pH 7.0, 0.2 mM EDTA, 20 μM phosphorylated GS2 peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole), GSK3b (5-20 mU, phosphorylated GS2 peptide (YRRAAVPPSPSLSRHSSPHQS(PO4)EDEEE), diluted to 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01% Brij35, 5% glycerol, 0.1% b-mercaptoethanol, 1 mg/ Ml BSA) and incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 19. ROCK-II (ROKa) test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、30 µM Long S6底物肽、10 mM乙酸鎂和0.02 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於Long S6底物肽（KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK）的ROCK-II (ROKa)（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。20. AMPK 試驗 Determined in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 30 μM Long S6 substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) ROCK-II (ROKa) for the Long S6 substrate peptide (KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK) (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 1 mg/ml BSA), Incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 20. AMPK test
在含有50 mM Hepes pH 7.5、1 mM DTT、0.02% Brij35、0.4 mM SAMS肽、0.196 mM AMP、10 mM乙酸鎂和0.05 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於SAMS底物肽（HMRSAMSGLHLVKRR）的AMPK（5-20 mU，稀釋於50 mM Hepes pH 7.5、1 mM DTT、0.02% Brij35中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。21. CHK1 試驗 25.5 μl containing 50 mM Hepes pH 7.5, 1 mM DTT, 0.02% Brij35, 0.4 mM SAMS peptide, 0.196 mM AMP, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) In the final volume, AMPK (5-20 mU, diluted in 50 mM Hepes pH 7.5, 1 mM DTT, 0.02% Brij35) against the SAMS substrate peptide (HMRSAMSGLHLVKRR) was assayed and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 21. CHK1 test
在含有8 mM MOPS pH 7.0、0.2 mM EDTA、200 µM CHKtide、10 mM乙酸鎂和0.02 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於CHKtide底物肽（KKKVSRSGLYRSPSMPENLNRPR）的CHK1（5-20 mU，稀釋於20 mM MOPS pH 7.5、1 mM EDTA、0.1%b-巰基乙醇、0.01% Brij-35、5%甘油、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。22. CK2 試驗 Determination of the bottom of CHKtide in a final volume of 25.5 μl containing 8 mM MOPS pH 7.0, 0.2 mM EDTA, 200 μM CHKtide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) CHK1 (5-20 mU of peptide peptide (KKKVSRSGLYRSPSMPENLNRPR) diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.1% b-mercaptoethanol, 0.01% Brij-35, 5% glycerol, 1 mg/ml BSA) Incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 22. CK2 test
在含有20 mM Hepes pH 7.5、0.15 M NaCl、0.1 mM EDTA、5 mM DTT、0.1% Triton-X 100、CKII肽（0.165 mM）、10 mM乙酸鎂和0.005 mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於CKII肽（RRRDDDSDDD）的CK2（5-20 mU，稀釋於20 mM Hepes pH7.5、0.15 M NaCl、0.1 mM EGTA、0.1% Triton X-100、5 mM DTT、50%甘油中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。23. PBK 試驗 Containing 20 mM Hepes pH 7.5, 0.15 M NaCl, 0.1 mM EDTA, 5 mM DTT, 0.1% Triton-X 100, CKII peptide (0.165 mM), 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (500 CK2 (5-20 mU for CKII peptide (RRRDDDSDDD), diluted to 20 mM Hepes pH 7.5, 0.15 M NaCl, 0.1 mM EGTA, 0.1% Triton X in a final volume of 25.5 μl at -1000 cpm/pmole) -100, 5 mM DTT, 50% glycerol) and incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 23. PBK test
在含有50 mM Tris pH 8.6、50 mMb-甘油磷酸鈉、0.04 mM CaCl2、磷酸化b肽（0.196 mM）、10 mM乙酸鎂、0.02 mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於磷酸化b肽（KRKQISVRGL）的PBK（5-20 mU，稀釋於50 mMb-甘油磷酸鈉 pH 7.0、0.1%b-巰基乙醇中），並在室溫下孵育15分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。24. LCK 試驗 Containing 50 mM Tris pH 8.6, 50 mMb-sodium glycerophosphate, 0.04 mM CaCl2, phosphorylated b-peptide (0.196 mM), 10 mM magnesium acetate, 0.02 mM [33P-g-ATP] (500-1000 cpm/pmole) PBK (5-20 mU, diluted in 50 mMb-sodium glycerophosphate pH 7.0, 0.1% b-mercaptoethanol) against the phosphorylated b-peptide (KRKQISVRGL) in a final volume of 25.5 μl and at room temperature Incubate for 15 minutes. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 24. LCK test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、0.1 mM Na3Vo4、Cdc2肽（0.25 mM）、10 mM乙酸鎂和0.05mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於Cdc2肽（KVEKIGEGTYGVVYK）的LCK（5-20 mU，稀釋於20 mM MOPS pH 7.5、1 mM EDTA、0.01% Brij35、5%甘油、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育15分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。25. CSK 試驗 25.5 μl final volume in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM Na3Vo4, Cdc2 peptide (0.25 mM), 10 mM magnesium acetate and 0.05 mM [33P-g-ATP] (500-1000 cpm/pmole) In the determination of LCK against Cdc2 peptide (KVEKIGEGTYGVVYK) (5-20 mU, diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01% Brij35, 5% glycerol, 0.1% b-mercaptoethanol, 1 mg/ml BSA Medium) and incubate for 15 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 25. CSK test
在含有8 mM MOPS pH7.0、0.2 mM EDTA、Cdc2肽（0.25 mM）、10 mM乙酸鎂和0.02 mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於Cdc2肽（KVEKIGEGTYGVVYK）的CSK（5-20 mU，稀釋於20 mM MOPS pH 7.5、1 mM EDTA、0.01% Brij35、5%甘油、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。26. CDK2/ 週期蛋白 A 試驗 Determined in a final volume of 25.5 μl containing 8 mM MOPS pH 7.0, 0.2 mM EDTA, Cdc2 peptide (0.25 mM), 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (500-1000 cpm/pmole) CSK against Cdc2 peptide (KVEKIGEGTYGVVYK) (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01% Brij35, 5% glycerol, 0.1% b-mercaptoethanol, 1 mg/ml BSA), Incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 26. CDK2/ cyclin A test
在含有50 mM Hepes pH7.5、1 mM DTT、0.02% Brij35、100 mM NaCl、組蛋白H1（1 mg/ml）、10 mM乙酸鎂和0.02 mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於組蛋白H1的CDK2/週期蛋白A（5-20 mU，稀釋於50 mM Hepes pH 7.5、1 mM DTT、0.02% Brij35、100 mM NaCl中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。27. DYRK 1A 試驗 Contains 50 mM Hepes pH 7.5, 1 mM DTT, 0.02% Brij35, 100 mM NaCl, histone H1 (1 mg/ml), 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (500-1000 cpm) CDK2/cyclin A (5-20 mU, diluted in 50 mM Hepes pH 7.5, 1 mM DTT, 0.02% Brij35, 100 mM NaCl) against histone H1 in a 25.5 μl final volume of /pmole) Incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 27. DYRK 1A test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、350 µM底物肽、10 mM乙酸鎂和0.05 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於Woodtide的DYRK 1A（5-20 mU，稀釋於50 mM Tris pH7.5、0.1 mM EGTA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。28. CK1 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 350 μM substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole), the assay was for Woodtide's DYRK 1A (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 28. CK1 test
在含有20 mM Hepes pH 7.5、0.15 M NaCl、0.1 mM EDTA、5 mM DTT、0.1% Triton-X 100、CKI肽（0.5 mM）、10 mM乙酸鎂和0.02 mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於CKI肽（RRKDLHDDEEDEAMSITA）的CK1（5-20 mU，稀釋於20 mM Hepes pH7.5、0.15 M NaCl、0.1 mM EGTA、0.1% Triton X-100、5 mM DTT、50%甘油中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。29. NEK6 試驗 Containing 20 mM Hepes pH 7.5, 0.15 M NaCl, 0.1 mM EDTA, 5 mM DTT, 0.1% Triton-X 100, CKI peptide (0.5 mM), 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (500 CK1 (5-20 mU for CKI peptide (RRKDLHDDEEDEAMSITA), diluted to 20 mM Hepes pH 7.5, 0.15 M NaCl, 0.1 mM EGTA, 0.1% Triton X in a final volume of 25.5 μl at -1000 cpm/pmole) -100, 5 mM DTT, 50% glycerol) and incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 29. NEK6 test
在含有50 mM Tris（pH 7.5）、0.1 mM EGTA、0.01% Brij、0.1%b-巰基乙醇、NEK6肽（0.3 mM）、10 mM乙酸鎂和0.05 mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於NEK6肽（FLAKSFGSPNRAYKK）的NEK6（5-20 mU，稀釋於50 mM Tris（pH 7.5）、0.1 mM EGTA、1 mg/ml BSA、0.1%b-巰基乙醇中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。30. NEK2a 試驗 Containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.01% Brij, 0.1% b-mercaptoethanol, NEK6 peptide (0.3 mM), 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (500-1000) NEK6 (5-20 mU, diluted to 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% b for NEK6 peptide (FLAKSFGSPNRAYKK) in a final volume of 25.5 μl of cpm/pmole) - mercaptoethanol) and incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 30. NEK2a test
在含有50 mM Tris（pH 7.5）、0.1 mM EGTA、0.01% Brij、0.1%b-巰基乙醇、300µM NEK2a肽、10 mM乙酸鎂和0.05 mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於NEK2a肽（RFRRSRRMI）的5-20 mU的NEK2a（稀釋於50 mM Tris（pH 7.5）、0.1 mM EGTA、1 mg/ml BSA、0.1%b-巰基乙醇中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。31. MAPKAP-K1b/RSK2 試驗 Containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.01% Brij, 0.1% b-mercaptoethanol, 300 μM NEK2a peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (500-1000 cpm/pmole) 5-20 mU of NEK2a against NEK2a peptide (RFRRSRRMI) in a final volume of 25.5 μl (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% b-mercaptoethanol) Medium) and incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 31. MAPKAP-K1b/RSK2 test
在含有50 mM b-甘油磷酸鈉（pH 7.5）、0.5 mM EDTA、30 µM底物肽、10 mM乙酸鎂和0.05 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（KKLNRTLSVA）的MAPKAP-K1b（5-20 mU，稀釋於20 mM MOPS pH 7.5、1 mM EDTA、0.01% Brij35、5%甘油、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。32. IKKb 試驗 At 25.5 μl final containing 50 mM b-glycerophosphate (pH 7.5), 0.5 mM EDTA, 30 μM substrate peptide, 10 mM magnesium acetate and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) In the volume, MAPKAP-K1b (5-20 mU, substrate diluted to 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01% Brij35, 5% glycerol, 0.1% b-mercaptoethanol, 1) was determined for the substrate peptide (KKLNRTLSVA). Mg/ml BSA) and incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 32. IKKb test
在含有50 mM Tris（pH 7.5）、0.1 mM EGTA、0.1%b-巰基乙醇、300µM底物肽、10 mM乙酸鎂和0.005 mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（LDDRHDSGLDSMKDEEY）的5-20 mU的IKKb（稀釋於50 mM Tris（pH 7.5）、0.1 mM EGTA、1 mg/ml BSA、0.1%b-巰基乙醇中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。33. smMLCK 試驗 25.5 containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% b-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (500-1000 cpm/pmole) In the final volume of μl, 5-20 mU of IKKb (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% b-mercaptoethanol) against the substrate peptide (LDDRHDSGLDSMKDEEY) was determined. And incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 33. smMLCK test
在含有50 mM Hepes（pH 7.5）、0.1 mM EGTA、5mM CaCl2、10µM鈣調素、300µM底物肽、10 mM乙酸鎂和0.05 mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（KKRPQRATSNVFA）的5-20 mU的smMLCK（稀釋於50 mM Hepes（pH 7.5）、0.1 mM EGTA、1 mg/mlBSA、0.1%b-巰基乙醇中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。34. PRK2 試驗 In a mixture containing 50 mM Hepes (pH 7.5), 0.1 mM EGTA, 5 mM CaCl2, 10 μM calmodulin, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (500-1000 cpm/pmole) 5-20 mU of smMLCK (diluted in 50 mM Hepes (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% b-mercaptoethanol) against the substrate peptide (KKRPQRATSNVFA) in a final volume of 25.5 μl And incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 34. PRK2 test
在含有50 mM Tris（pH 7.5）、0.1 mM EGTA、0.1%b-巰基乙醇、30µM Long S6肽、10 mM乙酸鎂和0.005 mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於Long S6肽（KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK）的5-20 mU的PRK2（稀釋於50 mM Tris（pH 7.5）、0.1 mM EGTA、1 mg/ml BSA、0.1%b-巰基乙醇中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。35. MNK2 ɑ試驗 25.5 containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% b-mercaptoethanol, 30 μM Long S6 peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (500-1000 cpm/pmole) 5-20 mU of PRK2 (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% b-mercaptoethanol) against the Long S6 peptide (KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK) in a final volume of μl And incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 35. MNK2 ɑ test
在含有50 mM Tris（pH 7.5）、0.1 mM EGTA、0.1%b-巰基乙醇、0.5 mg/ml底物肽、10 mM乙酸鎂和0.05 mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（eIF4E）的5-20 mU的MNK2（稀釋於50 mM Tris（pH 7.5）、0.1 mM EGTA、1 mg/ml BSA、0.1%b-巰基乙醇中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定。使用50 mM正磷酸的洗滌緩衝液將測定液收集到P81 Unifilter板上。36. CAMK-1 試驗 Contains 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% b-mercaptoethanol, 0.5 mg/ml substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (500-1000 cpm/pmole) 5-20 mU of MNK2 against substrate peptide (eIF4E) in a final volume of 25.5 μl (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% b-fluorenyl) In ethanol) and incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid. The assay solution was collected onto a P81 Unifilter plate using a wash buffer of 50 mM orthophosphoric acid. 36. CAMK-1 test
在含有50 mM Tris（pH 7.5）、0.1 mM EGTA、0.5mM CaCl2、0.3µM鈣調素、0.1%b-巰基乙醇、300µM底物肽、10 mM乙酸鎂和0.05 mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（YLRRRLSDSNF）的5-20 mU的CAMK-1（稀釋於50 mM Tris（pH 7.5）、0.1 mM EGTA、1 mg/ml BSA、0.1%b-巰基乙醇中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定。使用50 mM正磷酸的洗滌緩衝液將測定液收集到P81 Unifilter板上。37. PIM2 試驗 Containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.5 mM CaCl2, 0.3 μM calmodulin, 0.1% b-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] 5-20 mU of CAMK-1 (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg) against the substrate peptide (YLRRRLSDSNF) in a final volume of 25.5 μl (500-1000 cpm/pmole) /ml BSA, 0.1% b-mercaptoethanol) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid. The assay solution was collected onto a P81 Unifilter plate using a wash buffer of 50 mM orthophosphoric acid. 37. PIM2 test
在含有50 mM Tris（pH 7.5）、0.1 mM EGTA、0.5mM CaCl2、0.3µM鈣調素、0.1%b-巰基乙醇、300µM底物肽、10 mM乙酸鎂和0.005 mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（RSRHSSYPAGT）的5-20 mU的PIM2（稀釋於50 mM Tris（pH 7.5）、0.1 mM EGTA、1 mg/ml BSA、0.1%b-巰基乙醇中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定。使用50 mM正磷酸的洗滌緩衝液將測定液收集到P81 Unifilter板上。38. NEK7 試驗 Containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.5 mM CaCl2, 0.3 μM calmodulin, 0.1% b-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] 5-20 mU of PIM2 (diluted to 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml for the substrate peptide (RSRHSSYPAGT) in a final volume of 25.5 μl (500-1000 cpm/pmole) BSA, 0.1% b-mercaptoethanol) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid. The assay solution was collected onto a P81 Unifilter plate using a wash buffer of 50 mM orthophosphoric acid. 38. NEK7 test
在含有50 mM Tris（pH 7.5）、0.1 mM EGTA、0.01% Brij、0.1%b-巰基乙醇、底物肽(0.3 mM)、10 mM乙酸鎂和0.02 mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（FLAKSFGSPNRAYKK）的NEK7（5-20 mU，稀釋於50 mM Tris（pH 7.5）、0.1 mM EGTA、1 mg/ml BSA、0.1%b-巰基乙醇中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，且用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。39. JNK3 ɑ 1 試驗 Containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.01% Brij, 0.1% b-mercaptoethanol, substrate peptide (0.3 mM), 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (500- NEK7 (5-20 mU, diluted to 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1) against the substrate peptide (FLAKSFGSPNRAYKK) in a final volume of 25.5 μl at 1000 cpm/pmole) Incubate in %b-mercaptoethanol) and incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and collected on a P81 Unifilter plate with wash buffer of 50 mM orthophosphoric acid. 39. JNK3 ɑ 1 test
在25.5 µl終體積的50 mM Tris pH 7.5、0.1 mM EGTA、0.1% b-巰基乙醇、ATF2（3 µM）、10 mM乙酸鎂和0.05 mM[33P-g-ATP]（500-1000 cpm/pmole）中，測定針對於ATF2（啟動轉錄因數）的JNK3 ɑ1（5-20 mU，稀釋於50 mM Tris（pH 7.5）、0.1 mM EGTA、1 mg/ml BSA、0.1%b-巰基乙醇中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。40. MAPKAP-K3 試驗 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, ATF2 (3 μM), 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (500-1000 cpm/pmole) at a final volume of 25.5 μl In the assay, JNK3 ɑ1 (5-20 mU, diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% b-mercaptoethanol) against ATF2 (starting transcription factor), Incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 40. MAPKAP-K3 test
在含有50 mM Tris（pH 7.5）、0.1 mM EGTA、0.1%b-巰基乙醇、30µM底物肽、10 mM乙酸鎂和0.02 mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（KKLNRTLSVA）的5-20 mU的MAPKAP-K3（稀釋於50 mM Tris（pH 7.5）、0.1 mM EGTA、1 mg/ml BSA、0.1%b-巰基乙醇中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定。使用50 mM正磷酸的洗滌緩衝液將測定液收集到P81 Unifilter板上。41. ERK8 試驗 25.5 containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% b-mercaptoethanol, 30 μM substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (500-1000 cpm/pmole) 5-20 mU of MAPKAP-K3 (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% b-mercaptoethanol against the substrate peptide (KKLNRTLSVA) in a final volume of μl Medium) and incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid. The assay solution was collected onto a P81 Unifilter plate using a wash buffer of 50 mM orthophosphoric acid. 41. ERK8 test
在含有50 mM Tris（pH 7.5）、0.1 mM EGTA、0.1%b-巰基乙醇、0.33 mg/ml MBP、10 mM乙酸鎂和0.005 mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於MBP的5-20 mU的ERK8（稀釋於50 mM Tris（pH 7.5）、0.1 mM EGTA、1 mg/ml BSA、0.1%b-巰基乙醇中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定。使用50 mM正磷酸的洗滌緩衝液將測定液收集到P81 Unifilter板上。42. MNK1 試驗 In a mixture containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% b-mercaptoethanol, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (500-1000 cpm/pmole) In a final volume of 25.5 μl, determine 5-20 mU of ERK8 for MBP (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% b-mercaptoethanol) and in the chamber Incubate for 30 minutes at warm temperatures. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid. The assay solution was collected onto a P81 Unifilter plate using a wash buffer of 50 mM orthophosphoric acid. 42. MNK1 test
在含有50 mM Tris（pH 7.5）、0.1 mM EGTA、0.1%b-巰基乙醇、0.5 mg/ml底物肽、10 mM乙酸鎂和0.05 mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（eIF4E）的5-20 mU的MNK1（稀釋於50 mM Tris（pH 7.5）、0.1 mM EGTA、1 mg/ml BSA、0.1%b-巰基乙醇中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定。使用50 mM正磷酸的洗滌緩衝液將測定液收集到P81 Unifilter板上。43. SRPK1 試驗 Contains 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% b-mercaptoethanol, 0.5 mg/ml substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (500-1000 cpm/pmole) 5-20 mU of MNK1 (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% b-thiol) against the substrate peptide (eIF4E) in a final volume of 25.5 μl In ethanol) and incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid. The assay solution was collected onto a P81 Unifilter plate using a wash buffer of 50 mM orthophosphoric acid. 43. SRPK1 test
在含有50 mM Tris（pH 7.5）、0.1 mM EGTA、0.1%b-巰基乙醇、300µM底物肽、10 mM乙酸鎂和0.05 mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（RSRSRSRSRSRSRSR）的5-20 mU的SRPK1（稀釋於50 mM Tris（pH 7.5）、0.1 mM EGTA、1 mg/ml BSA、0.1%b-巰基乙醇中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定。使用50 mM正磷酸的洗滌緩衝液將測定液收集到P81 Unifilter板上。44. ΔPH-PKBβ(S474D) 試驗 25.5 containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% b-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (500-1000 cpm/pmole) In the final volume of μl, 5-20 mU of SRPK1 (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% b-mercaptoethanol) against the substrate peptide (RSRSRSRSRSRSRSR) was determined. And incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid. The assay solution was collected onto a P81 Unifilter plate using a wash buffer of 50 mM orthophosphoric acid. 44. ΔPH-PKBβ (S474D) test
在含有50 mM Tris pH 7.5、0.05%b-巰基乙醇、30 µM底物肽、10 mM乙酸鎂和0.05 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於經修飾的Crosstide肽（GRPRTSSFAEGKK）的ΔPH-PKB β-S474D（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。45. Aurora B 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% b-mercaptoethanol, 30 μM substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole), ΔPH-PKB β-S474D (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 1 mg/ml BSA) against the modified Crosstide peptide (GRPRTSSFAEGKK) was determined. And incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 45. Aurora B test
在含有50 mM Tris pH 7.5、0.05%b-巰基乙醇、300 µM底物肽、10 mM乙酸鎂和0.02 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（LRRLSLGLRRLSLGLRRLSLGLRRLSLG）的Aurora B（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。46. CHK2 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% b-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole), Determination of Aurora B (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 1 mg/ml BSA) against the substrate peptide (LRRLSLGLRRLSLGLRRLSLGLRRLSLG) at room temperature Incubate for 30 minutes. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 46. CHK2 test
在含有8 mM MOPS pH 7.0、0.2 mM EDTA、200 µM CHKtide、10 mM乙酸鎂和0.02 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於CHKtide底物肽（KKKVSRSGLYRSPSMPENLNRPR）的CHK2（5-20 mU，稀釋於20 mM MOPS pH 7.5、1 mM EDTA、0.1%b-巰基乙醇、0.01% Brij-35、5%甘油、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。47. Src 試驗 Determination of the bottom of CHKtide in a final volume of 25.5 μl containing 8 mM MOPS pH 7.0, 0.2 mM EDTA, 200 μM CHKtide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) Peptide peptide (KKKVSRSGLYRSPSMPENLNRPR) CHK2 (5-20 mU, diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.1% b-mercaptoethanol, 0.01% Brij-35, 5% glycerol, 1 mg/ml BSA), Incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 47. Src test
在含有50 mM Tris pH 7.5、0.05%b-巰基乙醇、300 µM底物肽、10 mM乙酸鎂和0.05 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（KVEKIGEGTYGVVYK）的Src（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。48. EF2K 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% b-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole), Src (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 1 mg/ml BSA) against the substrate peptide (KVEKIGEGTYGVVYK) was assayed and incubated at room temperature 30 minutes. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 48. EF2K test
在含有50 mM Hepes pH 6.6、0.2mM CaCl2、0.3µM鈣調素、0.05%b-巰基乙醇、300 µM底物肽、10 mM乙酸鎂和0.005 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（RKKFGESKTKTKEFL）的EF2K（5-20 mU，稀釋於50 mM Hepes pH 6.6、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。49. MARK3 試驗 Containing 50 mM Hepes pH 6.6, 0.2 mM CaCl2, 0.3 μM calmodulin, 0.05% b-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (50-1000 cpm) EF2K (5-20 mU, diluted in 50 mM Hepes pH 6.6, 0.1% b-mercaptoethanol, 1 mg/ml BSA) against the substrate peptide (RKKFGESKTKTKEFL) in a 25.5 μl final volume of /pmole) Incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 49. MARK3 test
在含有50 mM Tris pH 7.5、0.05%b-巰基乙醇、300 µM底物肽、10 mM乙酸鎂和0.005 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於CHKtide底物（KKKVSRSGLYRSPSMPENLNRPR）的MARK3（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。50. MST2 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% b-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole), MARK3 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 1 mg/ml BSA) against CHKtide substrate (KKKVSRSGLYRSPSMPENLNRPR) was assayed and incubated at room temperature 30 minutes. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 50. MST2 test
在含有50 mM Tris pH 7.5、0.05%b-巰基乙醇、0.33 mg/ml MBP、10 mM乙酸鎂和0.02 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於MBP的MST2（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1%b-巰基乙醇、100µM釩酸鹽中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。51. PKD1 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% b-mercaptoethanol, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole), MST2 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 100 μM vanadate) against MBP was assayed and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 51. PKD1 test
在含有50 mM Tris pH 7.5、0.05%b-巰基乙醇、30 µM底物肽、10 mM乙酸鎂和0.05 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（KKLNRTLSVA）的PKD1（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。52. PLK1 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% b-mercaptoethanol, 30 μM substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole), PKD1 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 1 mg/ml BSA) against the substrate peptide (KKLNRTLSVA) was assayed and incubated at room temperature 30 minutes. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 52. PLK1 test
在含有50 mM Tris pH 7.5、0.05%b-巰基乙醇、10µM釩酸鹽、300 µM底物肽、10 mM乙酸鎂和0.005 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（ISDELMDATFADQEAKKK）的PLK1（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1%b-巰基乙醇、1 mg/ml BSA、100µM釩酸鹽中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。53. DYRK2 試驗 25.5 containing 50 mM Tris pH 7.5, 0.05% b-mercaptoethanol, 10 μM vanadate, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) In the final volume of μl, PLK1 (5-20 mU, substrate diluted to 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 1 mg/ml BSA, 100 μM vanadic acid) was determined for the substrate peptide (ISDELMDATFADQEAKKK). Salt) and incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 53. DYRK2 test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、350 µM底物肽、10 mM乙酸鎂和0.05 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於Woodtide（KKISGRLSPIMTEQ）的DYRK2（5-20 mU，稀釋於50 mM Tris pH7.5、0.1 mM EGTA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。54. JNK2 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 350 μM substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole), the assay was for Woodtide (KKISGRLSPIMTEQ) DYRK2 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 54. JNK2 test
在25.5 µl終體積的50 mM Tris pH 7.5、0.1 mM EGTA、0.1% b-巰基乙醇、ATF2（3 µM）、10 mM乙酸鎂和0.02 mM[33P-g-ATP]（500-1000 cpm/pmole）中，測定針對於ATF2（啟動轉錄因數）的JNK2 1（5-20 mU，稀釋於50 mM Tris（pH 7.5）、0.1 mM EGTA、1 mg/ml BSA、0.1%b-巰基乙醇中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。55. DYRK3 試驗 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, ATF2 (3 μM), 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (500-1000 cpm/pmole) at a final volume of 25.5 μl In the assay, JNK2 1 (5-20 mU, diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% b-mercaptoethanol) against ATF2 (starting transcription factor), Incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 55. DYRK3 test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、350 µM底物肽、10 mM乙酸鎂和0.005 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於Woodtide（KKISGRLSPIMTEQ）的DYRK3（5-20 mU，稀釋於50 mM Tris pH7.5、0.1 mM EGTA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。56. HIPK2 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 350 μM substrate peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole), the assay was for Woodtide (KKISGRLSPIMTEQ) DYRK3 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 56. HIPK2 test
在含有50 mM Tris（pH 7.5）、0.1 mM EGTA、0.1%b-巰基乙醇、0.33 mg/ml MBP、10 mM乙酸鎂和0.005 mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於MBP的5-20 mU的HIPK2（稀釋於50 mM Tris（pH 7.5）、0.1 mM EGTA、1 mg/ml BSA、0.1%b-巰基乙醇中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。57. HIPK3 試驗 In a mixture containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% b-mercaptoethanol, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (500-1000 cpm/pmole) 5-20 mU of HIPK2 (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% b-mercaptoethanol) against MBP in a final volume of 25.5 μl, and in the chamber Incubate for 30 minutes at warm temperatures. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 57. HIPK3 test
在含有50 mM Tris（pH 7.5）、0.1 mM EGTA、0.1%b-巰基乙醇、0.33 mg/ml MBP、10 mM乙酸鎂和0.02 mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於MBP的5-20 mU的HIPK3（稀釋於50 mM Tris（pH 7.5）、0.1 mM EGTA、1 mg/ml BSA、0.1%b-巰基乙醇中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定。使用50 mM正磷酸的洗滌緩衝液將測定液收集到P81 Unifilter板上。58. PAK4 試驗 In a mixture containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% b-mercaptoethanol, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (500-1000 cpm/pmole) 5-20 mU of HIPK3 (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% b-mercaptoethanol) against MBP in a final volume of 25.5 μl, and in the chamber Incubate for 30 minutes at warm temperatures. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid. The assay solution was collected onto a P81 Unifilter plate using a wash buffer of 50 mM orthophosphoric acid. 58. PAK4 test
在含有50 mM Tris pH 7.5、0.05%b-巰基乙醇、300 µM底物肽、10 mM乙酸鎂和0.005 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（RRRLSFAEPG）的PAK4（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。59. PAK5 （ PAK7 ）試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% b-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole), PAK4 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 1 mg/ml BSA) against the substrate peptide (RRRLSFAEPG) was assayed and incubated at room temperature 30 minutes. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 59. PAK5 ( PAK7 ) test
在含有50 mM Tris pH 7.5、0.05%b-巰基乙醇、300 µM底物肽、10 mM乙酸鎂和0.02 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（RRRLSFAEPG）的PAK5（PAK7）（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。60. PAK6 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% b-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole), Determination of PAK5 (PAK7) against substrate peptide (RRRLSFAEPG) (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 1 mg/ml BSA), and in the chamber Incubate for 30 minutes at warm temperatures. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 60. PAK6 test
在含有50 mM Tris pH 7.5、0.05%b-巰基乙醇、300 µM底物肽、10 mM乙酸鎂和0.02 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（RRRLSFAEPG）的PAK6（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。61. CAMKKa 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% b-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole), PAK6 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 1 mg/ml BSA) against the substrate peptide (RRRLSFAEPG) was assayed and incubated at room temperature 30 minutes. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 61. CAMKKa test
在含有50 mM Tris（pH 7.5）、0.1 mM EGTA、0.5mM CaCl2、0.3µM鈣調素、0.1%b-巰基乙醇、300µM底物肽、10 mM乙酸鎂和0.02 mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（AKPKGNKDYHLQTCCGSLAYRRR）的5-20 mU的CAMKKa（稀釋於50 mM Tris（pH 7.5）、0.1 mM EGTA、1 mg/ml BSA、0.1%b-巰基乙醇中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定。使用50 mM正磷酸的洗滌緩衝液將測定液收集到P81 Unifilter板上。62. CAMKKb 試驗 Containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.5 mM CaCl2, 0.3 μM calmodulin, 0.1% b-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] 5-20 mU of CAMKKa (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml) against the substrate peptide (AKPKGNKDYHLQTCCGSLAYRRR) in a final volume of 25.5 μl (500-1000 cpm/pmole) BSA, 0.1% b-mercaptoethanol) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid. The assay solution was collected onto a P81 Unifilter plate using a wash buffer of 50 mM orthophosphoric acid. 62. CAMKKb test
在含有50 mM Tris（pH 7.5）、0.1 mM EGTA、0.5mM CaCl2、0.3µM鈣調素、0.1%b-巰基乙醇、300µM底物肽、10 mM乙酸鎂和0.02 mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（DGEFLRTSCGSPNYAARRR）的5-20 mU的CAMKKb（稀釋於50 mM Tris（pH 7.5）、0.1 mM EGTA、1 mg/ml BSA、0.1%b-巰基乙醇中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定。使用50 mM正磷酸的洗滌緩衝液將測定液收集到P81 Unifilter板上。63. PIM1 試驗 Containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.5 mM CaCl2, 0.3 μM calmodulin, 0.1% b-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] 5-20 mU of CAMKKb (diluted to 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml for the substrate peptide (DGEFLRTSCGSPNYAARRR) in a final volume of 25.5 μl (500-1000 cpm/pmole) BSA, 0.1% b-mercaptoethanol) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid. The assay solution was collected onto a P81 Unifilter plate using a wash buffer of 50 mM orthophosphoric acid. 63. PIM1 test
在含有50 mM Tris pH 7.5、0.05%b-巰基乙醇、300 µM底物肽、10 mM乙酸鎂和0.02 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（RSRHSSYPAGT）的PIM1（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。64. PIM3 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% b-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole), PIM1 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 1 mg/ml BSA) against the substrate peptide (RSRHSSYPAGT) was assayed and incubated at room temperature 30 minutes. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 64. PIM3 test
在含有50 mM Tris pH 7.5、0.05%b-巰基乙醇、300 µM底物肽、10 mM乙酸鎂和0.02 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（RSRHSSYPAGT）的PIM3（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。65. PLK1 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% b-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole), PIM3 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 1 mg/ml BSA) against the substrate peptide (RSRHSSYPAGT) was assayed and incubated at room temperature 30 minutes. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 65. PLK1 test
在含有50 mM Tris pH 7.5、0.05%b-巰基乙醇、10µM釩酸鹽、300 µM底物肽、10 mM乙酸鎂和0.005 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（ISDELMDATFADQEAKKK）的PLK1（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1%b-巰基乙醇、1 mg/ml BSA、100µM釩酸鹽中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。66. BRSK2 試驗 25.5 containing 50 mM Tris pH 7.5, 0.05% b-mercaptoethanol, 10 μM vanadate, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) In the final volume of μl, PLK1 (5-20 mU, substrate diluted to 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 1 mg/ml BSA, 100 μM vanadic acid) was determined for the substrate peptide (ISDELMDATFADQEAKKK). Salt) and incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 66. BRSK2 test
在含有50 mM Tris pH 7.5、0.05%b-巰基乙醇、300 µM底物肽、10 mM乙酸鎂和0.05 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（KKLNRTLSFAEPG）的BRSK2（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。67. MELK 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% b-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole), Determination of BRSK2 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 1 mg/ml BSA) against the substrate peptide (KKLNRTLSFAEPG) and incubating at room temperature 30 minutes. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 67. MELK test
在含有50 mM Tris pH 7.5、0.05%b-巰基乙醇、200 µM底物肽、10 mM乙酸鎂和0.05 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（KKLNRTLSFAEPG）的MELK（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。68. PKCζ 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% b-mercaptoethanol, 200 μM substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole), MELK (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 1 mg/ml BSA) against the substrate peptide (KKLNRTLSFAEPG) was assayed and incubated at room temperature 30 minutes. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 68. PKCζ test
在含有50 mM Tris pH 7.5、0.05%b-巰基乙醇、10µM釩酸鹽、300 µM底物肽、10 mM乙酸鎂和0.005 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（ERMRPRKRQGSVRRRV）的PKCζ（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1%b-巰基乙醇、1 mg/ml BSA、100µM釩酸鹽中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。69. Aurora C 試驗 25.5 containing 50 mM Tris pH 7.5, 0.05% b-mercaptoethanol, 10 μM vanadate, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) PKCζ (5-20 mU for substrate peptide (ERMRPRKRQGSVRRRV), diluted to 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 1 mg/ml BSA, 100 μM vanadic acid in a final volume of μl Salt) and incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 69. Aurora C test
在含有50 mM Tris pH 7.5、0.05%b-巰基乙醇、300 µM底物肽、10 mM乙酸鎂和0.005 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（LRRLSLGLRRLSLGLRRLSLGLRRLSLG）的Aurora C（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1%b-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。70. ERK1 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% b-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole), Determination of Aurora C (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 1 mg/ml BSA) against the substrate peptide (LRRLSLGLRRLSLGLRRLSLGLRRLSLG) at room temperature Incubate for 30 minutes. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 70. ERK1 test
在含有50 mM Tris（pH 7.5）、0.1 mM EGTA、0.1%b-巰基乙醇、0.33 mg/ml MBP、10 mM乙酸鎂和0.005 mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於MBP的5-20 mU 的ERK1（稀釋於50 mM Tris（pH 7.5）、0.1 mM EGTA、1 mg/ml BSA、0.1%b-巰基乙醇中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定。使用50 mM正磷酸的洗滌緩衝液將測定液收集到P81 Unifilter板上。71. FGF-R1 試驗 In a mixture containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% b-mercaptoethanol, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (500-1000 cpm/pmole) In a final volume of 25.5 μl, measure 5-20 mU of ERK1 against MBP (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% b-mercaptoethanol) and in the chamber Incubate for 30 minutes at warm temperatures. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid. The assay solution was collected onto a P81 Unifilter plate using a wash buffer of 50 mM orthophosphoric acid. 71. FGF-R1 test
在含有50 mM Tris pH 7.5、1 mg/ml底物肽、10 mM乙酸鎂和0.02 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（聚Glut Tyr）的FGF-R1（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。72. IRR 試驗 Determination of substrate for substrate in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 1 mg/ml substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) FGF-R1 (5-20 mU of peptide (poly Glut Tyr) diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 72. IRR test
在含有50 mM Hepes（pH 7.5）、0.1 mM EGTA、0.33 mg/ml MBP、10 mM乙酸鎂和0.005 mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於MBP的5-20 mU的IRR（稀釋於50 mM Hepes（pH 7.5）、0.1 mM EGTA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定。使用50 mM正磷酸的洗滌緩衝液將測定液收集到P81 Unifilter板上。73. EPH-A2 試驗 Determined in a final volume of 25.5 μl containing 50 mM Hepes (pH 7.5), 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (500-1000 cpm/pmole) IRR of 5-20 mU against MBP (diluted in 50 mM Hepes (pH 7.5), 0.1 mM EGTA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid. The assay solution was collected onto a P81 Unifilter plate using a wash buffer of 50 mM orthophosphoric acid. 73. EPH-A2 test
在含有50 mM Tris pH 7.5、0.1 mg/ml底物肽、10 mM乙酸鎂和0.05 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（聚Glut Tyr）的EPH-A2（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。74. MST4 試驗 Determination of substrate for substrate in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mg/ml substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) Peptide (poly Glut Tyr) EPH-A2 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 74. MST4 test
在含有50 mM Tris（pH 7.5）、0.1 mM EGTA、0.33 mg/ml MBP、10 mM乙酸鎂和0.02 mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於MBP的5-20 mU的MST4（稀釋於50 mM Tris（pH 7.5）、0.1 mM EGTA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定。使用50 mM正磷酸的洗滌緩衝液將測定液收集到P81 Unifilter板上。75. SYK 試驗 Determined in a final volume of 25.5 μl containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (500-1000 cpm/pmole) MST4 of 5-20 mU against MBP (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA) was incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid. The assay solution was collected onto a P81 Unifilter plate using a wash buffer of 50 mM orthophosphoric acid. 75. SYK test
在含有50 mM Tris pH 7.5、1 mg/ml底物肽、10 mM乙酸鎂和0.02 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（聚Glut Tyr）的SYK（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。76. YES1 試驗 Determination of substrate for substrate in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 1 mg/ml substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) Peptide (poly Glut Tyr) SYK (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 76. YES1 test
在含有50 mM Tris pH 7.5、1 mg/ml底物肽、10 mM乙酸鎂和0.02 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（聚Glut Tyr）的YES1（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。77. IGF-1R 試驗 Determination of substrate for substrate in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 1 mg/ml substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) The peptide (poly Glut Tyr) was YES1 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 77. IGF-1R test
在含有50 mM Tris pH 7.5、300µM底物肽、10 mM乙酸鎂和0.005 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（KKKSPGEYVNIEFG）的IGF-1R（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。78. VEG-FR 試驗 Determination of substrate peptide (KKKSPGEYVNIEFG) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 300 μM substrate peptide, 10 mM magnesium acetate and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) IGF-1R (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 78. VEG-FR test
在含有50 mM Tris pH 7.5、300µM底物肽、10 mM乙酸鎂和0.02 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（KKKSPGEYVNIEFG）的VEG-FR（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。79. BTK 試驗 Determination of substrate peptide (KKKSPGEYVNIEFG) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 300 μM substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) VEG-FR (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 79. BTK test
在含有50 mM Tris pH 7.5、300µM底物肽、10 mM乙酸鎂和0.05 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（KVEKIGEGTYGVVYK）的BTK（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。80. IR-HIS 試驗 Determination of substrate peptide (KVEKIGEGTYGVVYK) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 300 μM substrate peptide, 10 mM magnesium acetate and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) BTK (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 80. IR-HIS test
在含有50 mM Tris pH 7.5、300µM底物肽、10 mM乙酸鎂和0.02 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（KKSRGDYMTMQIG）的IR-HIS（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。81. EPH-B3 試驗 Determination of substrate peptide (KKSRGDYMTMQIG) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 300 μM substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) IR-HIS (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 81. EPH-B3 test
在含有50 mM Tris pH 7.5、1 mg/ml底物肽、10 mM乙酸鎂和0.005 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（聚Glut Tyr）的EPH-B3（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。82. TBK1 （ DU12569 ）試驗 Determination of substrate for substrate in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 1 mg/ml substrate peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) Peptide (poly Glut Tyr) EPH-B3 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 82. TBK1 ( DU12569 ) test
在含有50 mM Tris pH 7.5、300µM底物肽、10 mM乙酸鎂和0.05 mM[33P-g-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（KKKKERLLDDRHDSGLDSMKDEE）的TBK1（DU12569）（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。83. IKKepsilon （ DU14231 ）試驗 Determination of substrate peptide (KKKKERLLDDRHDSGLDSMKDEE) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 300 μM substrate peptide, 10 mM magnesium acetate and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) TBK1 (DU12569) (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 83. IKKepsilon ( DU14231 ) test
在含有50 mM Tris（pH 7.5）、0.1 mM EGTA、0.33 mg/ml MBP、10 mM乙酸鎂和0.05 mM[33P-g-ATP]（500-1000 cpm/pmole）的25.5 µl終體積中，測定針對於MBP的5-20 mU的IKKepsilon（DU14231）（稀釋於50 mM Tris（pH 7.5）、0.1 mM EGTA、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定。使用50 mM正磷酸的洗滌緩衝液將測定液收集到P81 Unifilter板上。84. GCK 試驗 Determined in a final volume of 25.5 μl containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (500-1000 cpm/pmole) IKKepsilon (DU14231) for 5-20 mU of MBP (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid. The assay solution was collected onto a P81 Unifilter plate using a wash buffer of 50 mM orthophosphoric acid. 84. GCK test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、0.33 mg/ml MBP、10 mM乙酸鎂和0.02mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於MBP的GCK（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1% β-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。85. IRAK4 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole), the assay was for MBP GCK (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 85. IRAK4 test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、0.33 mg/ml MBP、10 mM乙酸鎂和0.02mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於MBP的IRAK4（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1% β-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。86. NUAK1 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole), the assay was for MBP of IRAK4 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 86. NUAK1 test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、0.3mM ALNRTSSDSALHRRR、10 mM乙酸鎂和0.02mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於ALNRTSSDSALHRRR的NUAK1（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1% β-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。87. MLK1 試驗 Determination of ALNRTSSDSALHRRR in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.3 mM ALNRTSSDSALHRRR, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) NUAK1 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 87. MLK1 test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、0.33 mg/ml MBP、10 mM乙酸鎂和0.02mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於MBP的MLK1（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1% β-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。88. MINK1 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole), the assay was for MBP MLK1 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) was incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 88. MINK1 test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、0.33 mg/ml MBP、10 mM乙酸鎂和0.05mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於MBP的MINK1（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1% β-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。89. MLK3 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.05 mM [33P-γ-ATP] (50-1000 cpm/pmole), the assay was for MBP of MINK1 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 89. MLK3 test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、0.33 mg/ml MBP、10 mM乙酸鎂和0.02mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於MBP的MLK3（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1% β-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。90. LKB1 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole), the assay was for MBP of MLK3 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) was incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 90. LKB1 test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、0.2mM LSNLYHQGKFLQTFCGSPLYRRR、10 mM乙酸鎂和0.02mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於LSNLYHQGKFLQTFCGSPLYRRR的LKB1（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1% β-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。91. HER4 試驗 Determination of LSNLYHQGKFLQTFCGSPLYRRR in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.2 mM LSNLYHQGKFLQTFCGSPLYRRR, 10 mM magnesium acetate and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) LKB1 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 91. HER4 test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、1 mg/ml聚Glut Tyr、10 mM乙酸鎂和0.005mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於聚Glut Tyr的HER4（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1% β-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。92. TTK 試驗 Determined in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml poly Glut Tyr, 10 mM magnesium acetate, and 0.005 mM [33P-γ-ATP] (50-1000 cpm/pmole) HER4 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) against poly Glut Tyr was incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 92. TTK test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、0.3mM RSRSRSRSRSRSRSR、10 mM乙酸鎂和0.02mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於RSRSRSRSRSRSRSR的TTK（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1% β-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。93. RIPK2 試驗 Determination of the RSSRSRSRSRSRSRR in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.3 mM RSRSRSRSRSRSRSR, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) TTK (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 93. RIPK2 test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、0.33 mg/ml MBP、10 mM乙酸鎂和0.02mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於MBP的RIPK2（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1% β-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。94. Aurora A 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole), the assay was for MBP of RIPK2 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 94. Aurora A test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、0.3mM LRRLSLGLRRLSLGLRRLSLGLRRLSLG、10 mM乙酸鎂和0.005mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於LRRLSLGLRRLSLGLRRLSLGLRRLSLG的Aurora A（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1% β-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。95. PAK2 試驗 Determination of LRRLSLGLRRLSLGLRRLSLGLRRLSLG in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.3 mM LRRLSLGLRRLSLGLRRLSLGLRRLSLG, 10 mM magnesium acetate and 0.005 mM [33P-γ-ATP] (50-1000 cpm/pmole) Aurora A (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 95. PAK2 test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、0.3mM RRRLSFAEPG、10 mM乙酸鎂和0.02mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於RRRLSFAEPG的PAK2（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1% β-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。96. BRSK1 試驗 Determination of RRRLSFAEPG for a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.3 mM RRRLSFAEPG, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) PAK2 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 96. BRSK1 test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、0.3mM KKLNRTLSFAEPG、10 mM乙酸鎂和0.02mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於KKLNRTLSFAEPG的BRSK1（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1% β-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。97. HIPK3 試驗 Determination of KKLNRTLSFAEPG in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.3 mM KKLNRTLSFAEPG, 10 mM magnesium acetate and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) BRSK1 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 97. HIPK3 test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、0.33 mg/ml MBP、10 mM乙酸鎂和0.02mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於MBP的HIPK3（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1% β-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。98. HIPK1 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole), the assay was for MBP HIPK3 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 98. HIPK1 test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、0.33 mg/ml MBP、10 mM乙酸鎂和0.02mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於MBP的HIPK1（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1% β-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。99. JNK3α1 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole), the assay was for MBP HIPK1 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 99. JNK3α1 test
在25.5 µl終體積的50 mM Tris pH 7.5、0.1 mM EGTA、0.1% β-巰基乙醇、ATF2（3 µM）、10 mM乙酸鎂和0.02 mM[33P-γ−ATP] (500-1000 cpm/pmole)中，測定針對於ATF2（啟動轉錄因數）的JNK3（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、1 mg/ml BSA、0.1% β-巰基乙醇中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。100. MAPKAP-K3 試驗 Final volume of 25.5 μl of 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, ATF2 (3 μM), 10 mM magnesium acetate, and 0.02 mM [33P-γ−ATP] (500-1000 cpm/pmole) In the assay, JNK3 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA, 0.1% β-mercaptoethanol) against ATF2 (starting transcription factor), and in the room Incubate for 30 minutes at warm temperatures. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 100. MAPKAP-K3 test
在含有50 mM β-甘油磷酸鈉pH 7.5、0.5 mM EDTA、30 µM底物肽、10 mM乙酸鎂和0.02 mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於KKLNRTLSVA的MAPKAP-K3（5-20 mU，稀釋於20 mM MOPS pH 7.5、1 mM EDTA、0.01% Brij35、5%甘油、0.1% β-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。101. MARK2 試驗 In a final volume of 25.5 μl containing 50 mM sodium β-glycerophosphate pH 7.5, 0.5 mM EDTA, 30 μM substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) , MAPKAP-K3 (5-20 mU, diluted to 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01% Brij35, 5% glycerol, 0.1% β-mercaptoethanol, 1 mg/ml BSA) against KKLNRTLSVA, Incubate for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 101. MARK2 test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、0.3mM KKKVSRSGLYRSPSMPENLNRPR、10 mM乙酸鎂和0.02mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於KKKVSRSGLYRSPSMPENLNRPR的MARK2（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1% β-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。102. MARK4 試驗 Determination of KKKVSRSGLYRSPSMPENLNRPR in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.3 mM KKKVSRSGLYRSPSMPENLNRPR, 10 mM magnesium acetate and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) MARK2 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 102. MARK4 test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、0.3mM KKKVSRSGLYRSPSMPENLNRPR、10 mM乙酸鎂和0.05mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於KKKVSRSGLYRSPSMPENLNRPR的MARK4（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1% β-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。103. EPH-B4 試驗 Determination of KKKVSRSGLYRSPSMPENLNRPR for a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.3 mM KKKVSRSGLYRSPSMPENLNRPR, 10 mM magnesium acetate, and 0.05 mM [33P-γ-ATP] (50-1000 cpm/pmole) MARK4 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 103. EPH-B4 test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、10mM MnCl、1 mg/ml底物肽、10 mM乙酸鎂和0.05 mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（聚Glut Tyr）的EPH-B4（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、10mM MnCl、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。104. JAK2 試驗 25.5 μl final volume in 50 mM Tris pH 7.5, 0.1 mM EGTA, 10 mM MnCl, 1 mg/ml substrate peptide, 10 mM magnesium acetate and 0.05 mM [33P-γ-ATP] (50-1000 cpm/pmole) In the assay, EPH-B4 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 10 mM MnCl, 1 mg/ml BSA) against the substrate peptide (poly Glut Tyr) was measured at room temperature. Incubate for 30 minutes. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 104. JAK2 test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、0.05% β-巰基乙醇、100 µM底物肽、10mM乙酸鎂和0.005 mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於PDKtide（KTFCGTPEYLAPEVRREPRILSEEEQ-EMFRDFDYIADWC）的JAK2（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.05% β-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。105. EPH-A4 試驗 At 25.5 μl final containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.05% β-mercaptoethanol, 100 μM substrate peptide, 10 mM magnesium acetate, and 0.005 mM [33P-γ-ATP] (50-1000 cpm/pmole) In the volume, JAK2 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.05% β-mercaptoethanol, 1 mg/ml BSA) against PDKtide (KTFCGTPEYLAPEVRREPRILSEEEQ-EMFRDFDYIADWC) was determined and placed in the chamber. Incubate for 30 minutes at warm temperatures. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 105. EPH-A4 test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、1 mg/ml底物肽、10 mM乙酸鎂和0.05 mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（聚Glut Tyr）的EPH-A4（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、10mM MnCl、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。106. TAK1 試驗 Determined in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-γ-ATP] (50-1000 cpm/pmole) EPH-A4 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 10 mM MnCl, 1 mg/ml BSA) against the substrate peptide (poly Glut Tyr) and incubated at room temperature 30 minute. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 106. TAK1 test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、0.1% β-巰基乙醇、300µM底物肽、10 mM乙酸鎂、0.5mM MnCl和0.005 mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（RLGRDKYKTLRQIRQ）的TAK1（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。107. TrkA 試驗 Containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, 0.5 mM MnCl, and 0.005 mM [33P-γ-ATP] (50-1000 cpm/pmole) In a final volume of 25.5 μl, TAK1 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) against the substrate peptide (RLGRDKYKTLRQIRQ) was assayed and incubated at room temperature 30 minutes. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 107. TrkA test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、1 mg/ml底物肽、10 mM乙酸鎂和0.02 mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（聚Glut Tyr）的TrkA（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、10mM MnCl、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。108. MEKK1 試驗 Determined in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) TrkA (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 10 mM MnCl, 1 mg/ml BSA) against the substrate peptide (poly Glut Tyr) was incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 108. MEKK1 test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、0.33 mg/ml MBP、10 mM乙酸鎂和0.02mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於MBP的MEKK1（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1% β-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。109. MARK1 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole), the assay was for MBP of MEKK1 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 109. MARK1 test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、0.3mM KKKVSRSGLYRSPSMPENLNRPR、10 mM乙酸鎂和0.02mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於KKKVSRSGLYRSPSMPENLNRPR的MARK1（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1% β-巰基乙醇、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。110. CLK2 試驗 Determination of KKKVSRSGLYRSPSMPENLNRPR in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.3 mM KKKVSRSGLYRSPSMPENLNRPR, 10 mM magnesium acetate and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) MARK1 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 110. CLK2 test
在含有50 mM Tris pH 7.5、0.3mM肽、10mM DTT、10 mM乙酸鎂和0.005 mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（RNRYRDVSPFDHSR）的CLK2（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。111. DAPK1 試驗 Determination of substrate peptides in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.3 mM peptide, 10 mM DTT, 10 mM magnesium acetate, and 0.005 mM [33P-γ-ATP] (50-1000 cpm/pmole) (RNRYRDVSPFDHSR) CLK2 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 111. DAPK1 test
在含有50 mM Tris pH 7.5、0.3mM肽、10mM DTT、10 mM乙酸鎂和0.005 mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（KKLNRTLSFAEPG）的DAPK1（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。112. EPH-B2 試驗 Determination of substrate peptides in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.3 mM peptide, 10 mM DTT, 10 mM magnesium acetate, and 0.005 mM [33P-γ-ATP] (50-1000 cpm/pmole) (KKLNRTLSFAEPG) DAPK1 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 112. EPH-B2 test
在含有50 mM Tris pH 7.5、1 mg/ml底物肽、10 mM乙酸鎂和0.02 mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於底物肽（聚Glut Tyr）的EPH-B2（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、1 mg/ml BSA中），並在室溫下孵育30分鐘。通過加入5 μl的0.5 M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。113. TSSK1 試驗 Determination of substrate for substrate in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 1 mg/ml substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) Peptide (poly Glut Tyr) EPH-B2 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid, which was then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 113. TSSK1 test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、0.3 mM KKKVSRSGLYRSPSMPENLNRPR、10 mM乙酸鎂和0.02 mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於KKKVSRSGLYRSPSMPENLNRPR的TSSK1（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1% β-巰基乙醇、1 mg/ml BSA、10 mM DTT中），並在室溫下孵育30分鐘。通過加入5 μl的0.5 M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。114. TESK1 試驗 Determination of KKKVSRSGLYRSPSMPENLNRPR for a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.3 mM KKKVSRSGLYRSPSMPENLNRPR, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) TSSK1 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA, 10 mM DTT) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid, which was then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 114. TESK1 test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、0.2 mg/ml肌動蛋白素2、10 mM乙酸鎂和0.05 mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於肌動蛋白素2的TESK1（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1% β-巰基乙醇、1 mg/ml BSA、10MM DTT中），並在室溫下孵育30分鐘。通過加入5 μl的0.5 M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。115. TTBK1 試驗 In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.2 mg/ml actin 2, 10 mM magnesium acetate and 0.05 mM [33P-γ-ATP] (50-1000 cpm/pmole) , measuring TESK1 for actin 2 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA, 10 MM DTT), and in the room Incubate for 30 minutes at warm temperatures. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid, which was then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. 115. TTBK1 test
在含有50 mM Tris pH 7.5、0.1 mM EGTA、0.3mM RRKDLHDDEEDEAMSITA、10 mM乙酸鎂和0.005 mM[33P-γ-ATP]（50-1000 cpm/pmole）的25.5 µl終體積中，測定針對於RRKDLHDDEEDEAMSITA的TTBK1（5-20 mU，稀釋於50 mM Tris pH 7.5、0.1 mM EGTA、0.1% β-巰基乙醇、1 mg/ml BSA、10 mM DTT中），並在室溫下孵育30分鐘。通過加入5 μl的0.5 M（3%）正磷酸來停止測定，隨後用50 mM正磷酸的洗滌緩衝液收集到P81 Unifilter板上。LRRK2 效力 Determination of RRKDLHDDEEDEAMSITA for a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.3 mM RRKDLHDDEEDEAMSITA, 10 mM magnesium acetate and 0.005 mM [33P-γ-ATP] (50-1000 cpm/pmole) TTBK1 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA, 10 mM DTT) and incubated for 30 minutes at room temperature. The assay was stopped by the addition of 5 μl of 0.5 M (3%) orthophosphoric acid, which was then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid. LRRK2 effectiveness
下表示出了以本發明實施例抑制LRRK2 G2019S的IC50
值。表 實施例的 LRRK2_IC50 值 
下表示出了本發明實施例的JAK2 IC50
值。表 實施例的 JAK2_IC50 值 
下表示出了本發明實施例的存活率。
**P＜0.01，與DMSO陰性對照相比； *P＜0.05，與DMSO陰性對照相比。攀爬試驗 **P < 0.01 compared to DMSO negative control; *P < 0.05 compared to DMSO negative control. Climbing test
下表示出了本發明實施例的攀爬測定。
**P＜0.01，與DMSO陰性對照相比； *P＜0.05，與DMSO陰性對照相比；在第 6 周進行的激酶試驗 ** P <0.01, compared with DMSO negative control; * P <0.05, compared to the negative control DMSO; kinase assays performed in 6 weeks
下表示出了本發明實施例的激酶測定結果。
*P＜0.05，與DMSO陰性對照相比。激酶選擇性數據 *P < 0.05 compared to DMSO negative controls. Kinase selectivity data
代表性化合物的激酶選擇性資料如下表所示。以1μM抑制劑濃度下的每種特異性激酶的抑制百分比表示值。表 代表性化合物的激酶選擇性資料 
在不脫離本發明的範圍和精神下，本發明描述方面的各種修改和變體對於本領域技術人員將是顯而易見的。雖然已經結合具體的較佳實施方式描述了本發明，但是應當理解，所要求保護的本發明不應被不適當地限於這些具體實施方式。實際上，實施本發明的所描述的模式的各種修改，這種修改對於相關領域的技術人員是顯而易見的，意圖在所附權利要求的範圍內。參考文獻 Various modifications and variations of the described aspects of the invention will be apparent to those skilled in the art. Although the present invention has been described in connection with the preferred embodiments thereof, it is understood that the claimed invention should not be In fact, various modifications of the described modes of the invention are apparent to those skilled in the art and are intended to be within the scope of the appended claims. references
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